U.S. patent application number 17/225866 was filed with the patent office on 2021-10-21 for crispr-cas effector polypeptides and methods of use thereof.
The applicant listed for this patent is The Regents of the University of California. Invention is credited to Basem Al-Shayeb, Jillian F. Banfield, Jennifer A. Doudna, Patrick Pausch.
Application Number | 20210324356 17/225866 |
Document ID | / |
Family ID | 1000005735425 |
Filed Date | 2021-10-21 |
United States Patent
Application |
20210324356 |
Kind Code |
A1 |
Doudna; Jennifer A. ; et
al. |
October 21, 2021 |
CRISPR-CAS EFFECTOR POLYPEPTIDES AND METHODS OF USE THEREOF
Abstract
The present disclosure provides RNA-guided CRISPR-Cas effector
proteins, nucleic acids encoding same, and compositions comprising
same. The present disclosure provides ribonucleoprotein complexes
comprising: an RNA-guided CRISPR-Cas effector protein of the
present disclosure; and a guide RNA. The present disclosure
provides methods of modifying a target nucleic acid, using an
RNA-guided CRISPR-Cas effector protein of the present disclosure
and a guide RNA. The present disclosure provides methods of
modulating transcription of a target nucleic acid.
Inventors: |
Doudna; Jennifer A.;
(Berkeley, CA) ; Al-Shayeb; Basem; (Berkeley,
CA) ; Banfield; Jillian F.; (Berkeley, CA) ;
Pausch; Patrick; (Berkeley, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Family ID: |
1000005735425 |
Appl. No.: |
17/225866 |
Filed: |
April 8, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2020/021213 |
Mar 5, 2020 |
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17225866 |
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62948470 |
Dec 16, 2019 |
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62907422 |
Sep 27, 2019 |
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62855739 |
May 31, 2019 |
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62815173 |
Mar 7, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2800/80 20130101;
C07K 2319/09 20130101; C12N 15/11 20130101; A61K 31/7088 20130101;
C12N 15/907 20130101; A61K 38/465 20130101; C12N 2310/20 20170501;
C12N 9/22 20130101 |
International
Class: |
C12N 9/22 20060101
C12N009/22; C12N 15/11 20060101 C12N015/11; C12N 15/90 20060101
C12N015/90; A61K 38/46 20060101 A61K038/46; A61K 31/7088 20060101
A61K031/7088 |
Claims
1.-149. (canceled)
150. A composition comprising: a) a nuclease comprising a single
RuvC active site capable of both cleaving DNA and binding crRNA and
wherein the amino terminus (N terminus) of the nuclease does not
begin with the amino acid sequence "MIS"; and b) a recombinant
guide RNA.
151. The composition of claim 150, wherein the N terminus of the
nuclease does not begin with the amino acid sequence "MISK".
152. The composition of claim 150, wherein the nuclease comprises
an amino acid sequence that is at least 50% identical to SEQ ID NO:
120.
153. The composition of claim 150, wherein the recombinant guide
RNA comprises a nucleobase sequence that is at least 80% identical
to SEQ ID NO: 15 or 139.
154. The composition of claim 150, wherein the recombinant guide
RNA comprises a nucleobase sequence that is at least 95% identical
to SEQ ID NO: 15 or 139.
155. The composition of claim 150, comprising a nuclear
localization signal (NLS) that is fused to the N terminus of the
nuclease, the C terminus of the nuclease, or both termini of the
nuclease.
156. The composition of claim 155, wherein the NLS is fused
directly to the N terminus of the nuclease via an amide bond, and
wherein the NLS comprises the amino acid sequence set forth in SEQ
ID NO: 49.
157. The composition of claim 155, wherein the NLS is fused to the
carboxyl terminus (C terminus) of the nuclease, and wherein the NLS
comprises the amino acid sequence set forth in SEQ ID NO: 50.
158. The composition of claim 155, wherein a first NLS comprising
the amino acid sequence set forth in SEQ ID NO: 49 is fused to the
N terminus of the nuclease via an amide bond and wherein a second
NLS comprising the amino acid sequence set forth in SEQ ID NO: 50
is fused to the C terminus of the nuclease.
159. The composition of claim 150, wherein the nuclease is a
nickase or a catalytically inactive nuclease.
160. The composition of claim 150, comprising a DNA donor template,
an additional recombinant guide RNA, or a combination thereof.
161. The composition of claim 150, wherein the nuclease recognizes
a TTN protospacer adjacent motif, wherein T is thymine and N is any
nucleotide.
162. The composition of claim 150, comprising a lipid, a liposome,
a vector, or a particle.
163. The composition of claim 150, comprising one or more of: a
buffer, a nuclease inhibitor, and a protease inhibitor.
164. A pharmaceutical composition comprising the composition of
claim 150 and a pharmaceutically acceptable excipient.
165. The composition of claim 150, comprising a detectable
label.
166. A method of editing a gene comprising contacting a cell
comprising the gene with the composition of claim 150.
167. A population of cells modified by the composition of claim
150.
168. A method of producing a recombinant protein, comprising
culturing the population of cells of claim 167.
169. A cell comprising the composition of claim 150.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 62/815,173, filed Mar. 7, 2019, U.S.
Provisional Patent Application No. 62/855,739, filed May 31, 2019,
U.S. Provisional Patent Application No. 62/907,422, filed Sep. 27,
2019, and U.S. Provisional Patent Application No. 62/948,470, filed
Dec. 16, 2019, each of which applications is incorporated herein by
reference in its entirety.
INTRODUCTION
[0002] CRISPR-Cas systems include Cas proteins, which are involved
in acquisition, targeting and cleavage of foreign DNA or RNA, and a
guide RNA(s), which includes a segment that binds Cas proteins and
a segment that binds to a target nucleic acid. For example, Class 2
CRISPR-Cas systems comprise a single Cas protein bound to a guide
RNA, where the Cas protein binds to and cleaves a targeted nucleic
acid. The programmable nature of these systems has facilitated
their use as a versatile technology for use in modification of
target nucleic acid.
SUMMARY
[0003] The present disclosure provides RNA-guided CRISPR-Cas
effector proteins, nucleic acids encoding same, and compositions
comprising same. The present disclosure provides ribonucleoprotein
complexes comprising: an RNA-guided CRISPR-Cas effector protein of
the present disclosure; and a guide RNA. The present disclosure
provides methods of modifying a target nucleic acid, using an
RNA-guided CRISPR-Cas effector protein of the present disclosure
and a guide RNA. The present disclosure provides methods of
modulating transcription of a target nucleic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1A shows the size distribution of complete
bacteriophage genomes from this study, Lak phage reported recently
from a subset of the same samples and reference sources (all dsDNA
genomes from RefSeq v92 and non-artifactual assemblies >200 kb
from (Paez-Espino et al. (2016) Nature 536: 425).
[0005] FIG. 1B shows a histogram of the genome size distribution of
phage with genomes >200 kb from this study, Lak, and reference
genomes. Box and whisker plots of tRNA counts per genome as a
function of genome size.
[0006] FIG. 2 shows a phylogenetic tree constructed using terminase
sequences from huge phage genomes of this study and related
database sequences. Colored regions of the tree indicate large
clades of phage, all of which have huge genomes.
[0007] FIG. 3 shows a model for how phage-encoded capacities could
function to redirect the host's translational system to produce
phage proteins. No huge phage has all of these genes, but many have
tRNAs (clover leaf shapes) and tRNA synthetases (aaRS). Phage
proteins with up to 6 ribosomal protein S1 domains occur in a few
genomes. The S1 binds mRNA to bring it into the site on the
ribosome where it is decoded. Ribosomal protein S21 (S21) might
selectively initiate translation of phage mRNAs, and many sequences
have N-terminal extensions that may be involved in binding RNA
(dashed line in ribosome insert, which is based on PDB code 6bu8
and pmid: 29247757 for ribosome and S1 structural model). Some
phage have initiation factors (IF) and elongation factor G (EF G)
and some have rpL7/L12, which could mediate efficient ribosome
binding. Abbreviation: RNA pol, RNA polymerase.
[0008] FIG. 4A shows a bacterium-phage interaction involving CRISPR
targeting (cell diagram).
[0009] FIG. 4B shows the interaction network showing targeting of
bacterial (from top to bottom: SEQ ID NOs: 163-164) and
phage-encoded (from top to bottom: SEQ ID NOs: 163-164) CRISPR
spacers.
[0010] FIG. 5 shows ecosystems with phage and some plasmids with
>200 kbp genomes, grouped by sampling site type. Each box
represents a phage genome, and boxes are arranged in order of
decreasing genome size; size range for each site type is listed to
the right. Colors indicate putative host phylum based on genome
phylogenetic profile, with confirmation by CRISPR targeting (X) or
information system gene phylogenetic analyses (T).
[0011] FIG. 6A-6R provide amino acid sequences of examples of
Cas12J polypeptides of the present disclosure.
[0012] FIG. 7 provides nucleotide sequences of constant region
portions of Cas12J guide RNAs (Depicted as the DNA encoding the
RNA). Sequences in bold are the orientation used and/or
extrapolated from the working examples (see, e.g., the crRNA
`sequences used` in Example 3). Sequences separated by an "or" are
the reverse complement of one another.
[0013] FIG. 8 depicts consensus sequences for Cas12J guide
RNAs.
[0014] FIG. 9 provides the positions of amino acids in RuvC-I,
RuvC-II, and RuvC-III domains of Cas12J polypeptides that, when
substituted, results in a Cas12J polypeptide that binds, but does
not cleave, a target nucleic acid in the presence of a Cas12J guide
RNA.
[0015] FIG. 10 provides a tree showing various CRISPR-Cas effector
protein families.
[0016] FIG. 11A-11C shows the efficiency of transformation plasmid
interference assay.
[0017] FIG. 12A-12B shows a demonstration that Cas12J (e.g.,
Cas12J-1947455, Cas12J-2071242 and Cas12J-3339380) can cleave
linear dsDNA fragments guided by a crRNA spacer sequence.
[0018] FIG. 13 shows results demonstrating the elucidation of PAM
sequences.
[0019] FIG. 14A-14C illustrates results from mapping RNA sequences
to the Cas12J CRISPR loci from pBAS::Cas12J-1947455,
pBAS::Cas12J-2071242, and pBAS::Cas12J-3339380.
[0020] FIG. 15 depicts Cas12j-2- and Cas12j-3-mediated gene editing
in human cells.
[0021] FIG. 16A-16B provide maps of the pCas12J-3-hs (FIG. 16A) and
pCas12J-2-hs (FIG. 16B) constructs.
[0022] FIG. 17A-17G present Table 1, which provides nucleotide
sequences of the pCas12J-2-hs and pCas12J-3-hs constructs (from top
to bottom: SEQ ID NOs: 161-162).
[0023] FIG. 18 depicts trans cleavage of ssDNA by Cas12J activated
by binding to DNA.
[0024] FIG. 19A-19F depict data showing that Cas12J (Cas.PHI.) is a
bona fide CRISPR-Cas system.
[0025] FIG. 20 presents a maximum likelihood phylogenetic tree of
type V subtypes a-k.
[0026] FIG. 21A-21B present crRNA repeat similarity (FIG. 21A)
among various Cas12J crRNAs and Cas12J amino acid sequence identity
(FIG. 21B) among various Cas12J proteins.
[0027] FIG. 22A-22C depict Cas.PHI.-3-mediated protection against
plasmid transformation.
[0028] FIG. 23A-23D depict cleavage of DNA by Cas.PHI..
[0029] FIG. 24A-24D depict purification of apo Cas.PHI. (Cas.PHI.
protein without guide RNA).
[0030] FIG. 25A-25C depict production of staggered cuts by
Cas.PHI..
[0031] FIG. 26A-26B depict Cas.PHI.-mediated cleavage of dsDNA and
ssDNA.
[0032] FIG. 27A-27B depict the results of a cleavage assay
comparing target strand (TS) and non-target strand (NTS) cleavage
efficiency by Cas.PHI..
[0033] FIG. 28A-28B depict data showing that Cas.PHI. cleaves
ssDNA, but not RNA, in trans upon activation in cis.
[0034] FIG. 29A-29D depict processing of pre-crRNA by Cas.PHI.
within the RuvC active site.
[0035] FIG. 30A-30C depict processing of pre-crRNA by Cas.PHI.-1
and by Cas.PHI.-2.
[0036] FIG. 31A-31B depict formation of ribonucleoprotein (RNP)
complexes with: a) pre-crRNA
[0037] FIG. 32A-32C depict Cas.PHI.-mediated enhanced green
fluorescent protein (EGFP) disruption in HEK293 cells.
[0038] FIG. 33A-33B depict data showing Cas.PHI.-mediate genome
editing in human cells.
[0039] FIG. 34 presents Table 3, which provides a description of
some of the plasmids used in Example 7.
[0040] FIG. 35 presents Table 4, which provides guide sequences for
experiments described in Example 7.
[0041] FIG. 36 presents Table 5, which provides substrate sequences
for in vitro experiments described in Example 7.
[0042] FIG. 37 presents Table 6, which provides crRNA sequences for
in vitro experiments described in Example 7.
DEFINITIONS
[0043] The terms "polynucleotide" and "nucleic acid," used
interchangeably herein, refer to a polymeric form of nucleotides of
any length, either ribonucleotides or deoxyribonucleotides. Thus,
this term includes, but is not limited to, single-, double-, or
multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a
polymer comprising purine and pyrimidine bases or other natural,
chemically or biochemically modified, non-natural, or derivatized
nucleotide bases.
[0044] By "hybridizable" or "complementary" or "substantially
complementary" it is meant that a nucleic acid (e.g. RNA, DNA)
comprises a sequence of nucleotides that enables it to
non-covalently bind, i.e. form Watson-Crick base pairs and/or G/U
base pairs, "anneal", or "hybridize," to another nucleic acid in a
sequence-specific, antiparallel, manner (i.e., a nucleic acid
specifically binds to a complementary nucleic acid) under the
appropriate in vitro and/or in vivo conditions of temperature and
solution ionic strength. Standard Watson-Crick base-pairing
includes: adenine (A) pairing with thymidine (T), adenine (A)
pairing with uracil (U), and guanine (G) pairing with cytosine (C)
[DNA, RNA]. In addition, for hybridization between two RNA
molecules (e.g., dsRNA), and for hybridization of a DNA molecule
with an RNA molecule (e.g., when a DNA target nucleic acid base
pairs with a guide RNA, etc.): guanine (G) can also base pair with
uracil (U). For example, G/U base-pairing is at least partially
responsible for the degeneracy (i.e., redundancy) of the genetic
code in the context of tRNA anti-codon base-pairing with codons in
mRNA. Thus, in the context of this disclosure, a guanine (G) (e.g.,
of dsRNA duplex of a guide RNA molecule; of a guide RNA base
pairing with a target nucleic acid, etc.) is considered
complementary to both a uracil (U) and to an adenine (A). For
example, when a G/U base-pair can be made at a given nucleotide
position of a dsRNA duplex of a guide RNA molecule, the position is
not considered to be non-complementary, but is instead considered
to be complementary.
[0045] Hybridization and washing conditions are well known and
exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T.
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor (1989), particularly
Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell,
W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor (2001). The
conditions of temperature and ionic strength determine the
"stringency" of the hybridization.
[0046] Hybridization requires that the two nucleic acids contain
complementary sequences, although mismatches between bases are
possible. The conditions appropriate for hybridization between two
nucleic acids depend on the length of the nucleic acids and the
degree of complementarity, variables well known in the art. The
greater the degree of complementarity between two nucleotide
sequences, the greater the value of the melting temperature (Tm)
for hybrids of nucleic acids having those sequences. For
hybridizations between nucleic acids with short stretches of
complementarity (e.g. complementarity over 35 or less, 30 or less,
25 or less, 22 or less, 20 or less, or 18 or less nucleotides) the
position of mismatches can become important (see Sambrook et al.,
supra, 11.7-11.8). Typically, the length for a hybridizable nucleic
acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12
nucleotides or more, 15 nucleotides or more, 20 nucleotides or
more, 22 nucleotides or more, 25 nucleotides or more, or 30
nucleotides or more). Temperature, wash solution salt
concentration, and other conditions may be adjusted as necessary
according to factors such as length of the region of
complementation and the degree of complementation.
[0047] It is understood that the sequence of a polynucleotide need
not be 100% complementary to that of its target nucleic acid to be
specifically hybridizable or hybridizable. Moreover, a
polynucleotide may hybridize over one or more segments such that
intervening or adjacent segments are not involved in the
hybridization event (e.g., a bulge, a loop structure or hairpin
structure, etc.). A polynucleotide can comprise 60% or more, 65% or
more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or
more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100%
sequence complementarity to a target region within the target
nucleic acid sequence to which it will hybridize. For example, an
antisense nucleic acid in which 18 of 20 nucleotides of the
antisense compound are complementary to a target region, and would
therefore specifically hybridize, would represent 90 percent
complementarity. In this example, the remaining noncomplementary
nucleotides may be clustered or interspersed with complementary
nucleotides and need not be contiguous to each other or to
complementary nucleotides. Percent complementarity between
particular stretches of nucleic acid sequences within nucleic acids
can be determined using any convenient method. Example methods
include BLAST programs (basic local alignment search tools) and
PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215,
403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), the Gap
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, Madison Wis.),
e.g., using default settings, which uses the algorithm of Smith and
Waterman (Adv. Appl. Math., 1981, 2, 482-489), and the like.
[0048] The terms "peptide," "polypeptide," and "protein" are used
interchangeably herein, and refer to a polymeric form of amino
acids of any length, which can include coded and non-coded amino
acids, chemically or biochemically modified or derivatized amino
acids, and polypeptides having modified peptide backbones.
[0049] "Binding" as used herein (e.g. with reference to an
RNA-binding domain of a polypeptide, binding to a target nucleic
acid, and the like) refers to a non-covalent interaction between
macromolecules (e.g., between a protein and a nucleic acid; between
a Cas12J polypeptide/guide RNA complex and a target nucleic acid;
and the like). While in a state of non-covalent interaction, the
macromolecules are said to be "associated" or "interacting" or
"binding" (e.g., when a molecule X is said to interact with a
molecule Y, it is meant the molecule X binds to molecule Y in a
non-covalent manner). Not all components of a binding interaction
need be sequence-specific (e.g., contacts with phosphate residues
in a DNA backbone), but some portions of a binding interaction may
be sequence-specific. Binding interactions are generally
characterized by a dissociation constant (K.sub.D) of less than
10.sup.-6 M, less than 10.sup.-7 M, less than 10.sup.-8 M, less
than 10.sup.-9 M, less than 10.sup.-11 M, less than 10.sup.-11 M,
less than 10.sup.-12 M, less than 10.sup.-13 M, less than
10.sup.-14 M, or less than 10.sup.-15 M. "Affinity" refers to the
strength of binding, increased binding affinity being correlated
with a lower K.sub.D.
[0050] By "binding domain" it is meant a protein domain that is
able to bind non-covalently to another molecule. A binding domain
can bind to, for example, a DNA molecule (a DNA-binding domain), an
RNA molecule (an RNA-binding domain) and/or a protein molecule (a
protein-binding domain). In the case of a protein having a
protein-binding domain, it can in some cases bind to itself (to
form homodimers, homotrimers, etc.) and/or it can bind to one or
more regions of a different protein or proteins.
[0051] The term "conservative amino acid substitution" refers to
the interchangeability in proteins of amino acid residues having
similar side chains. For example, a group of amino acids having
aliphatic side chains consists of glycine, alanine, valine,
leucine, and isoleucine; a group of amino acids having
aliphatic-hydroxyl side chains consists of serine and threonine; a
group of amino acids having amide containing side chains consisting
of asparagine and glutamine; a group of amino acids having aromatic
side chains consists of phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains consists of lysine,
arginine, and histidine; a group of amino acids having acidic side
chains consists of glutamate and aspartate; and a group of amino
acids having sulfur containing side chains consists of cysteine and
methionine. Exemplary conservative amino acid substitution groups
are: valine-leucine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine-glycine, and
asparagine-glutamine.
[0052] A polynucleotide or polypeptide has a certain percent
"sequence identity" to another polynucleotide or polypeptide,
meaning that, when aligned, that percentage of bases or amino acids
are the same, and in the same relative position, when comparing the
two sequences. Sequence identity can be determined in a number of
different ways. To determine sequence identity, sequences can be
aligned using various convenient methods and computer programs
(e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the
world wide web at sites including ncbi.nlm.nili.gov/BLAST,
ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/,
mafft.cbrc.jp/alignment/software/. See, e.g., Altschul et al.
(1990), J. Mol. Bioi. 215:403-10.
[0053] A DNA sequence that "encodes" a particular RNA is a DNA
nucleotide sequence that is transcribed into RNA. A DNA
polynucleotide may encode an RNA (mRNA) that is translated into
protein (and therefore the DNA and the mRNA both encode the
protein), or a DNA polynucleotide may encode an RNA that is not
translated into protein (e.g. tRNA, rRNA, microRNA (miRNA), a
"non-coding" RNA (ncRNA), a guide RNA, etc.).
[0054] A "protein coding sequence" or a sequence that encodes a
particular protein or polypeptide, is a nucleotide sequence that is
transcribed into mRNA (in the case of DNA) and is translated (in
the case of mRNA) into a polypeptide in vitro or in vivo when
placed under the control of appropriate regulatory sequences.
[0055] The terms "DNA regulatory sequences," "control elements,"
and "regulatory elements," used interchangeably herein, refer to
transcriptional and translational control sequences, such as
promoters, enhancers, polyadenylation signals, terminators, protein
degradation signals, and the like, that provide for and/or regulate
transcription of a non-coding sequence (e.g., guide RNA) or a
coding sequence (e.g., RNA-guided endonuclease, GeoCas9
polypeptide, GeoCas9 fusion polypeptide, and the like) and/or
regulate translation of an encoded polypeptide.
[0056] As used herein, a "promoter" or a "promoter sequence" is a
DNA regulatory region capable of binding RNA polymerase and
initiating transcription of a downstream (3' direction) coding or
non-coding sequence. For purposes of the present disclosure, the
promoter sequence is bounded at its 3' terminus by the
transcription initiation site and extends upstream (5' direction)
to include the minimum number of bases or elements necessary to
initiate transcription at levels detectable above background.
Within the promoter sequence will be found a transcription
initiation site, as well as protein binding domains responsible for
the binding of RNA polymerase. Eukaryotic promoters will often, but
not always, contain "TATA" boxes and "CAT" boxes. Various
promoters, including inducible promoters, may be used to drive
expression by the various vectors of the present disclosure.
[0057] The term "naturally-occurring" or "unmodified" or "wild
type" as used herein as applied to a nucleic acid, a polypeptide, a
cell, or an organism, refers to a nucleic acid, polypeptide, cell,
or organism that is found in nature. For example, a polypeptide or
polynucleotide sequence that is present in an organism that can be
isolated from a source in nature is naturally occurring.
[0058] The term "fusion" as used herein as applied to a nucleic
acid or polypeptide refers to two components that are defined by
structures derived from different sources. For example, where
"fusion" is used in the context of a fusion polypeptide (e.g., a
fusion Cas12J protein), the fusion polypeptide includes amino acid
sequences that are derived from different polypeptides. A fusion
polypeptide may comprise either modified or naturally-occurring
polypeptide sequences (e.g., a first amino acid sequence from a
modified or unmodified Cas12J protein; and a second amino acid
sequence from a modified or unmodified protein other than a Cas12J
protein, etc.). Similarly, "fusion" in the context of a
polynucleotide encoding a fusion polypeptide includes nucleotide
sequences derived from different coding regions (e.g., a first
nucleotide sequence encoding a modified or unmodified Cas12J
protein; and a second nucleotide sequence encoding a polypeptide
other than a Cas12J protein).
[0059] The term "fusion polypeptide" refers to a polypeptide which
is made by the combination (i.e., "fusion") of two otherwise
separated segments of amino acid sequence, usually through human
intervention.
[0060] "Heterologous," as used herein, means a nucleotide or
polypeptide sequence that is not found in the native nucleic acid
or protein, respectively. For example, in some cases, in a variant
Cas12J protein of the present disclosure, a portion of
naturally-occurring Cas12J polypeptide (or a variant thereof) may
be fused to a heterologous polypeptide (i.e. an amino acid sequence
from a protein other than a Cas12J polypeptide or an amino acid
sequence from another organism). As another example, a fusion
Cas12J polypeptide can comprise all or a portion of a
naturally-occurring Cas12J polypeptide (or variant thereof) fused
to a heterologous polypeptide, i.e., a polypeptide from a protein
other than a Cas12J polypeptide, or a polypeptide from another
organism. The heterologous polypeptide may exhibit an activity
(e.g., enzymatic activity) that will also be exhibited by the
variant Cas12J protein or the fusion Cas12J protein (e.g., biotin
ligase activity; nuclear localization; etc.). A heterologous
nucleic acid sequence may be linked to a naturally-occurring
nucleic acid sequence (or a variant thereof) (e.g., by genetic
engineering) to generate a nucleotide sequence encoding a fusion
polypeptide (a fusion protein).
[0061] "Recombinant," as used herein, means that a particular
nucleic acid (DNA or RNA) is the product of various combinations of
cloning, restriction, polymerase chain reaction (PCR) and/or
ligation steps resulting in a construct having a structural coding
or non-coding sequence distinguishable from endogenous nucleic
acids found in natural systems. DNA sequences encoding polypeptides
can be assembled from cDNA fragments or from a series of synthetic
oligonucleotides, to provide a synthetic nucleic acid which is
capable of being expressed from a recombinant transcriptional unit
contained in a cell or in a cell-free transcription and translation
system. Genomic DNA comprising the relevant sequences can also be
used in the formation of a recombinant gene or transcriptional
unit. Sequences of non-translated DNA may be present 5' or 3' from
the open reading frame, where such sequences do not interfere with
manipulation or expression of the coding regions, and may indeed
act to modulate production of a desired product by various
mechanisms (see "DNA regulatory sequences"). Alternatively, DNA
sequences encoding RNA (e.g., guide RNA) that is not translated may
also be considered recombinant. Thus, e.g., the term "recombinant"
nucleic acid refers to one which is not naturally occurring, e.g.,
is made by the artificial combination of two otherwise separated
segments of sequence through human intervention. This artificial
combination is often accomplished by either chemical synthesis
means, or by the artificial manipulation of isolated segments of
nucleic acids, e.g., by genetic engineering techniques. Such is
usually done to replace a codon with a codon encoding the same
amino acid, a conservative amino acid, or a non-conservative amino
acid. Alternatively, it is performed to join together nucleic acid
segments of desired functions to generate a desired combination of
functions. This artificial combination is often accomplished by
either chemical synthesis means, or by the artificial manipulation
of isolated segments of nucleic acids, e.g., by genetic engineering
techniques. When a recombinant polynucleotide encodes a
polypeptide, the sequence of the encoded polypeptide can be
naturally occurring ("wild type") or can be a variant (e.g., a
mutant) of the naturally occurring sequence. An example of such a
case is a DNA (a recombinant) encoding a wild-type protein where
the DNA sequence is codon optimized for expression of the protein
in a cell (e.g., a eukaryotic cell) in which the protein is not
naturally found (e.g., expression of a CRISPR/Cas RNA-guided
polypeptide such as Cas12J (e.g., wild-type Cas12J; variant Cas12J;
fusion Cas12J; etc.) in a eukaryotic cell). A codon-optimized DNA
can therefore be recombinant and non-naturally occurring while the
protein encoded by the DNA may have a wild type amino acid
sequence.
[0062] Thus, the term "recombinant" polypeptide does not
necessarily refer to a polypeptide whose amino acid sequence does
not naturally occur. Instead, a "recombinant" polypeptide is
encoded by a recombinant non-naturally occurring DNA sequence, but
the amino acid sequence of the polypeptide can be naturally
occurring ("wild type") or non-naturally occurring (e.g., a
variant, a mutant, etc.). Thus, a "recombinant" polypeptide is the
result of human intervention, but may have a naturally occurring
amino acid sequence.
[0063] A "vector" or "expression vector" is a replicon, such as
plasmid, phage, virus, artificial chromosome, or cosmid, to which
another DNA segment, i.e. an "insert", may be attached so as to
bring about the replication of the attached segment in a cell.
[0064] An "expression cassette" comprises a DNA coding sequence
operably linked to a promoter. "Operably linked" refers to a
juxtaposition wherein the components so described are in a
relationship permitting them to function in their intended manner.
For instance, a promoter is operably linked to a coding sequence
(or the coding sequence can also be said to be operably linked to
the promoter) if the promoter affects its transcription or
expression.
[0065] The terms "recombinant expression vector," or "DNA
construct" are used interchangeably herein to refer to a DNA
molecule comprising a vector and an insert. Recombinant expression
vectors are usually generated for the purpose of expressing and/or
propagating the insert(s), or for the construction of other
recombinant nucleotide sequences. The insert(s) may or may not be
operably linked to a promoter sequence and may or may not be
operably linked to DNA regulatory sequences.
[0066] A cell has been "genetically modified" or "transformed" or
"transfected" by exogenous DNA or exogenous RNA, e.g. a recombinant
expression vector, when such DNA has been introduced inside the
cell. The presence of the exogenous DNA results in permanent or
transient genetic change. The transforming DNA may or may not be
integrated (covalently linked) into the genome of the cell. In
prokaryotes, yeast, and mammalian cells for example, the
transforming DNA may be maintained on an episomal element such as a
plasmid. With respect to eukaryotic cells, a stably transformed
cell is one in which the transforming DNA has become integrated
into a chromosome so that it is inherited by daughter cells through
chromosome replication. This stability is demonstrated by the
ability of the eukaryotic cell to establish cell lines or clones
that comprise a population of daughter cells containing the
transforming DNA. A "clone" is a population of cells derived from a
single cell or common ancestor by mitosis. A "cell line" is a clone
of a primary cell that is capable of stable growth in vitro for
many generations.
[0067] Suitable methods of genetic modification (also referred to
as "transformation") include e.g., viral or bacteriophage
infection, transfection, conjugation, protoplast fusion,
lipofection, electroporation, calcium phosphate precipitation,
polyethyleneimine (PEI)-mediated transfection, DEAE-dextran
mediated transfection, liposome-mediated transfection, particle gun
technology, calcium phosphate precipitation, direct micro
injection, nanoparticle-mediated nucleic acid delivery (see, e.g.,
Panyam et al. Adv Drug Deliv Rev. 2012 Sep. 13. pii:
S0169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023), and the
like.
[0068] The choice of method of genetic modification is generally
dependent on the type of cell being transformed and the
circumstances under which the transformation is taking place (e.g.,
in vitro, ex vivo, or in vivo). A general discussion of these
methods can be found in Ausubel, et al., Short Protocols in
Molecular Biology, 3rd ed., Wiley & Sons, 1995.
[0069] A "target nucleic acid" as used herein is a polynucleotide
(e.g., DNA such as genomic DNA) that includes a site ("target site"
or "target sequence") targeted by an RNA-guided endonuclease
polypeptide (e.g., wild-type Cas12J; variant Cas12J; fusion Cas12J;
etc.). The target sequence is the sequence to which the guide
sequence of a subject Cas12J guide RNA (e.g., a dual Cas12J guide
RNA or a single-molecule Cas12J guide RNA) will hybridize. For
example, the target site (or target sequence) 5'-GAGCAUAUC-3'
within a target nucleic acid is targeted by (or is bound by, or
hybridizes with, or is complementary to) the sequence
5'-GAUAUGCUC-3'. Suitable hybridization conditions include
physiological conditions normally present in a cell. For a double
stranded target nucleic acid, the strand of the target nucleic acid
that is complementary to and hybridizes with the guide RNA is
referred to as the "complementary strand" or "target strand"; while
the strand of the target nucleic acid that is complementary to the
"target strand" (and is therefore not complementary to the guide
RNA) is referred to as the "non-target strand" or
"non-complementary strand."
[0070] By "cleavage" it is meant the breakage of the covalent
backbone of a target nucleic acid molecule (e.g., RNA, DNA).
Cleavage can be initiated by a variety of methods including, but
not limited to, enzymatic or chemical hydrolysis of a
phosphodiester bond. Both single-stranded cleavage and
double-stranded cleavage are possible, and double-stranded cleavage
can occur as a result of two distinct single-stranded cleavage
events.
[0071] "Nuclease" and "endonuclease" are used interchangeably
herein to mean an enzyme which possesses catalytic activity for
nucleic acid cleavage (e.g., ribonuclease activity (ribonucleic
acid cleavage), deoxyribonuclease activity (deoxyribonucleic acid
cleavage), etc.).
[0072] By "cleavage domain" or "active domain" or "nuclease domain"
of a nuclease it is meant the polypeptide sequence or domain within
the nuclease which possesses the catalytic activity for nucleic
acid cleavage. A cleavage domain can be contained in a single
polypeptide chain or cleavage activity can result from the
association of two (or more) polypeptides. A single nuclease domain
may consist of more than one isolated stretch of amino acids within
a given polypeptide.
[0073] The term "stem cell" is used herein to refer to a cell
(e.g., plant stem cell, vertebrate stem cell) that has the ability
both to self-renew and to generate a differentiated cell type (see
Morrison et al. (1997) Cell 88:287-298). In the context of cell
ontogeny, the adjective "differentiated", or "differentiating" is a
relative term. A "differentiated cell" is a cell that has
progressed further down the developmental pathway than the cell it
is being compared with. Thus, pluripotent stem cells (described
below) can differentiate into lineage-restricted progenitor cells
(e.g., mesodermal stem cells), which in turn can differentiate into
cells that are further restricted (e.g., neuron progenitors), which
can differentiate into end-stage cells (i.e., terminally
differentiated cells, e.g., neurons, cardiomyocytes, etc.), which
play a characteristic role in a certain tissue type, and may or may
not retain the capacity to proliferate further. Stem cells may be
characterized by both the presence of specific markers (e.g.,
proteins, RNAs, etc.) and the absence of specific markers. Stem
cells may also be identified by functional assays both in vitro and
in vivo, particularly assays relating to the ability of stem cells
to give rise to multiple differentiated progeny.
[0074] Stem cells of interest include pluripotent stem cells
(PSCs). The term "pluripotent stem cell" or "PSC" is used herein to
mean a stem cell capable of producing all cell types of the
organism. Therefore, a PSC can give rise to cells of all germ
layers of the organism (e.g., the endoderm, mesoderm, and ectoderm
of a vertebrate). Pluripotent cells are capable of forming
teratomas and of contributing to ectoderm, mesoderm, or endoderm
tissues in a living organism. Pluripotent stem cells of plants are
capable of giving rise to all cell types of the plant (e.g., cells
of the root, stem, leaves, etc.).
[0075] PSCs of animals can be derived in a number of different
ways. For example, embryonic stem cells (ESCs) are derived from the
inner cell mass of an embryo (Thomson et. al, Science. 1998 Nov. 6;
282(5391):1145-7) whereas induced pluripotent stem cells (iPSCs)
are derived from somatic cells (Takahashi et. al, Cell. 2007 Nov.
30; 131(5):861-72; Takahashi et. al, Nat Protoc. 2007;
2(12):3081-9; Yu et. al, Science. 2007 Dec. 21; 318(5858):1917-20.
Epub 2007 Nov. 20). Because the term PSC refers to pluripotent stem
cells regardless of their derivation, the term PSC encompasses the
terms ESC and iPSC, as well as the term embryonic germ stem cells
(EGSC), which are another example of a PSC. PSCs may be in the form
of an established cell line, they may be obtained directly from
primary embryonic tissue, or they may be derived from a somatic
cell. PSCs can be target cells of the methods described herein.
[0076] By "embryonic stem cell" (ESC) is meant a PSC that was
isolated from an embryo, typically from the inner cell mass of the
blastocyst. ESC lines are listed in the NIH Human Embryonic Stem
Cell Registry, e.g. hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04
(BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell
International); Miz-hES1 (MizMedi Hospital-Seoul National
University); HSF-1, HSF-6 (University of California at San
Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research
Foundation (WiCell Research Institute)). Stem cells of interest
also include embryonic stem cells from other primates, such as
Rhesus stem cells and marmoset stem cells. The stem cells may be
obtained from any mammalian species, e.g. human, equine, bovine,
porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate,
etc. (Thomson et al. (1998) Science 282:1145; Thomson et al. (1995)
Proc. Natl. Acad. Sci USA 92:7844; Thomson et al. (1996) Biol.
Reprod. 55:254; Shamblott et al., Proc. Natl. Acad. Sci. USA
95:13726, 1998). In culture, ESCs typically grow as flat colonies
with large nucleo-cytoplasmic ratios, defined borders and prominent
nucleoli. In addition, ESCs express SSEA-3, SSEA-4, TRA-1-60,
TRA-1-81, and Alkaline Phosphatase, but not SSEA-1. Examples of
methods of generating and characterizing ESCs may be found in, for
example, U.S. Pat. Nos. 7,029,913, 5,843,780, and 6,200,806, the
disclosures of which are incorporated herein by reference. Methods
for proliferating hESCs in the undifferentiated form are described
in WO 99/20741, WO 01/51616, and WO 03/020920.
[0077] By "embryonic germ stem cell" (EGSC) or "embryonic germ
cell" or "EG cell" is meant a PSC that is derived from germ cells
and/or germ cell progenitors, e.g. primordial germ cells, i.e.
those that would become sperm and eggs. Embryonic germ cells (EG
cells) are thought to have properties similar to embryonic stem
cells as described above. Examples of methods of generating and
characterizing EG cells may be found in, for example, U.S. Pat. No.
7,153,684; Matsui, Y., et al., (1992) Cell 70:841; Shamblott, M.,
et al. (2001) Proc. Natl. Acad. Sci. USA 98: 113; Shamblott, M., et
al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U.,
et al. (1996) Development, 122:1235, the disclosures of which are
incorporated herein by reference.
[0078] By "induced pluripotent stem cell" or "iPSC" it is meant a
PSC that is derived from a cell that is not a PSC (i.e., from a
cell this is differentiated relative to a PSC). iPSCs can be
derived from multiple different cell types, including terminally
differentiated cells. iPSCs have an ES cell-like morphology,
growing as flat colonies with large nucleo-cytoplasmic ratios,
defined borders and prominent nuclei. In addition, iPSCs express
one or more key pluripotency markers known by one of ordinary skill
in the art, including but not limited to Alkaline Phosphatase,
SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b,
FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples of methods of
generating and characterizing iPSCs may be found in, for example,
U.S. Patent Publication Nos. US20090047263, US20090068742,
US20090191159, US20090227032, US20090246875, and US20090304646, the
disclosures of which are incorporated herein by reference.
Generally, to generate iPSCs, somatic cells are provided with
reprogramming factors (e.g. Oct4, SOX2, KLF4, MYC, Nanog, Lin28,
etc.) known in the art to reprogram the somatic cells to become
pluripotent stem cells.
[0079] By "somatic cell" it is meant any cell in an organism that,
in the absence of experimental manipulation, does not ordinarily
give rise to all types of cells in an organism. In other words,
somatic cells are cells that have differentiated sufficiently that
they will not naturally generate cells of all three germ layers of
the body, i.e. ectoderm, mesoderm and endoderm. For example,
somatic cells would include both neurons and neural progenitors,
the latter of which may be able to naturally give rise to all or
some cell types of the central nervous system but cannot give rise
to cells of the mesoderm or endoderm lineages.
[0080] By "mitotic cell" it is meant a cell undergoing mitosis.
Mitosis is the process by which a eukaryotic cell separates the
chromosomes in its nucleus into two identical sets in two separate
nuclei. It is generally followed immediately by cytokinesis, which
divides the nuclei, cytoplasm, organelles and cell membrane into
two cells containing roughly equal shares of these cellular
components.
[0081] By "post-mitotic cell" it is meant a cell that has exited
from mitosis, i.e., it is "quiescent", i.e. it is no longer
undergoing divisions. This quiescent state may be temporary, i.e.
reversible, or it may be permanent.
[0082] By "meiotic cell" it is meant a cell that is undergoing
meiosis. Meiosis is the process by which a cell divides its nuclear
material for the purpose of producing gametes or spores. Unlike
mitosis, in meiosis, the chromosomes undergo a recombination step
which shuffles genetic material between chromosomes. Additionally,
the outcome of meiosis is four (genetically unique) haploid cells,
as compared with the two (genetically identical) diploid cells
produced from mitosis.
[0083] In some instances, a component (e.g., a nucleic acid
component (e.g., a Cas12J guide RNA); a protein component (e.g.,
wild-type Cas12J polypeptide; variant Cas12J polypeptide; fusion
Cas12J polypeptide; etc.); and the like) includes a label moiety.
The terms "label", "detectable label", or "label moiety" as used
herein refer to any moiety that provides for signal detection and
may vary widely depending on the particular nature of the assay.
Label moieties of interest include both directly detectable labels
(direct labels; e.g., a fluorescent label) and indirectly
detectable labels (indirect labels; e.g., a binding pair member). A
fluorescent label can be any fluorescent label (e.g., a fluorescent
dye (e.g., fluorescein, Texas red, rhodamine, ALEXAFLUOR.RTM.
labels, and the like), a fluorescent protein (e.g., green
fluorescent protein (GFP), enhanced GFP (EGFP), yellow fluorescent
protein (YFP), red fluorescent protein (RFP), cyan fluorescent
protein (CFP), cherry, tomato, tangerine, and any fluorescent
derivative thereof), etc.). Suitable detectable (directly or
indirectly) label moieties for use in the methods include any
moiety that is detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical, chemical, or
other means. For example, suitable indirect labels include biotin
(a binding pair member), which can be bound by streptavidin (which
can itself be directly or indirectly labeled). Labels can also
include: a radiolabel (a direct label)(e.g., .sup.3H, .sup.125I,
.sup.35S, .sup.14C, or .sup.32P); an enzyme (an indirect
label)(e.g., peroxidase, alkaline phosphatase, galactosidase,
luciferase, glucose oxidase, and the like); a fluorescent protein
(a direct label)(e.g., green fluorescent protein, red fluorescent
protein, yellow fluorescent protein, and any convenient derivatives
thereof); a metal label (a direct label); a colorimetric label; a
binding pair member; and the like. By "partner of a binding pair"
or "binding pair member" is meant one of a first and a second
moiety, wherein the first and the second moiety have a specific
binding affinity for each other. Suitable binding pairs include,
but are not limited to: antigen/antibodies (for example,
digoxigenin/anti-digoxigenin, dinitrophenyl (DNP)/anti-DNP,
dansyl-X-anti-dansyl, fluorescein/anti-fluorescein, lucifer
yellow/anti-lucifer yellow, and rhodamine anti-rhodamine),
biotin/avidin (or biotin/streptavidin) and calmodulin binding
protein (CBP)/calmodulin. Any binding pair member can be suitable
for use as an indirectly detectable label moiety.
[0084] Any given component, or combination of components can be
unlabeled, or can be detectably labeled with a label moiety. In
some cases, when two or more components are labeled, they can be
labeled with label moieties that are distinguishable from one
another.
[0085] General methods in molecular and cellular biochemistry can
be found in such standard textbooks as Molecular Cloning: A
Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory
Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel
et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag
et al., John Wiley & Sons 1996); Nonviral Vectors for Gene
Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors
(Kaplift & Loewy eds., Academic Press 1995); Immunology Methods
Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue
Culture: Laboratory Procedures in Biotechnology (Doyle &
Griffiths, John Wiley & Sons 1998), the disclosures of which
are incorporated herein by reference.
[0086] As used herein, the terms "treatment," "treating," and the
like, refer to obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease or symptom thereof and/or may be
therapeutic in terms of a partial or complete cure for a disease
and/or adverse effect attributable to the disease. "Treatment," as
used herein, covers any treatment of a disease in a mammal, e.g.,
in a human, and includes: (a) preventing the disease from occurring
in a subject which may be predisposed to the disease but has not
yet been diagnosed as having it; (b) inhibiting the disease, i.e.,
arresting its development; and (c) relieving the disease, i.e.,
causing regression of the disease.
[0087] The terms "individual," "subject," "host," and "patient,"
used interchangeably herein, refer to an individual organism, e.g.,
a mammal, including, but not limited to, murines, simians, humans,
non-human primates, ungulates, felines, canines, bovines, ovines,
mammalian farm animals, mammalian sport animals, and mammalian
pets.
[0088] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0089] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0090] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0091] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a Cas12J CRISPR-Cas effector polypeptide"
includes a plurality of such polypeptides and reference to "the
guide RNA" includes reference to one or more guide RNAs and
equivalents thereof known to those skilled in the art, and so
forth. It is further noted that the claims may be drafted to
exclude any optional element. As such, this statement is intended
to serve as antecedent basis for use of such exclusive terminology
as "solely," "only" and the like in connection with the recitation
of claim elements, or use of a "negative" limitation.
[0092] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable sub-combination.
All combinations of the embodiments pertaining to the invention are
specifically embraced by the present invention and are disclosed
herein just as if each and every combination was individually and
explicitly disclosed. In addition, all sub-combinations of the
various embodiments and elements thereof are also specifically
embraced by the present invention and are disclosed herein just as
if each and every such sub-combination was individually and
explicitly disclosed herein.
[0093] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
DETAILED DESCRIPTION
[0094] The present disclosure provides RNA-guided CRISPR-Cas
effector proteins, referred to herein as "Cas12J" polypeptides,
"Cas.PHI." polypeptides, or "CasXS" polypeptides"; nucleic acids
encoding same; and compositions comprising same. The present
disclosure provides ribonucleoprotein complexes comprising: a
Cas12J polypeptide of the present disclosure; and a guide RNA. The
present disclosure provides methods of modifying a target nucleic
acid, using a Cas12J polypeptide of the present disclosure and a
guide RNA. The present disclosure provides methods of modulating
transcription of a target nucleic acid.
[0095] The present disclosure provides guide RNAs (referred to
herein as "Cas12J guide RNAs") that bind to and provide sequence
specificity to the Cas12J proteins; nucleic acids encoding the
Cas12J guide RNAs; and modified host cells comprising the Cas12J
guide RNAs and/or nucleic acids encoding same. Cas12J guide RNAs
are useful in a variety of applications, which are provided.
[0096] Compositions
[0097] CRISPR/CAS12J Proteins and Guide RNAs
[0098] A Cas12J CRISPR/Cas effector polypeptide (e.g., a Cas12J
protein; also referred to as a "CasXS polypeptide" or a "Cas.PHI.
polypeptide") interacts with (binds to) a corresponding guide RNA
(e.g., a Cas12J guide RNA) to form a ribonucleoprotein (RNP)
complex that is targeted to a particular site in a target nucleic
acid (e.g. a target DNA) via base pairing between the guide RNA and
a target sequence within the target nucleic acid molecule. A guide
RNA includes a nucleotide sequence (a guide sequence) that is
complementary to a sequence (the target site) of a target nucleic
acid. Thus, a Cas12J protein forms a complex with a Cas12J guide
RNA and the guide RNA provides sequence specificity to the RNP
complex via the guide sequence. The Cas12J protein of the complex
provides the site-specific activity. In other words, the Cas12J
protein is guided to a target site (e.g., stabilized at a target
site) within a target nucleic acid sequence (e.g. a chromosomal
sequence or an extrachromosomal sequence, e.g., an episomal
sequence, a minicircle sequence, a mitochondrial sequence, a
chloroplast sequence, etc.) by virtue of its association with the
guide RNA.
[0099] In some cases, a Cas12J CRISPR/Cas effector polypeptide of
the present disclosure, when complexed with a guide RNA, cleaves
double-stranded DNA or single-stranded DNA, but not single-stranded
RNA.
[0100] In some cases, a Cas12J CRISPR/Cas effector polypeptide of
the present disclosure catalyzes processing of pre-crRNA in a
magnesium-dependent manner.
[0101] The present disclosure provides compositions comprising a
Cas12J polypeptide (and/or a nucleic acid comprising a nucleotide
sequence encoding the Cas12J polypeptide) (e.g., where the Cas12J
polypeptide can be a naturally existing protein, a nickase Cas12J
protein, a catalytically inactive ("dead" Cas12J; also referred to
herein as a "dCas12J protein"), a fusion Cas12J protein, etc.). The
present disclosure provides compositions comprising a Cas12J guide
RNA (and/or a nucleic acid comprising a nucleotide sequence
encoding the Cas12J guide RNA). The present disclosure provides
compositions comprising (a) a Cas12J polypeptide (and/or a nucleic
acid encoding the Cas12J polypeptide) (e.g., where the Cas12J
polypeptide can be a naturally existing protein, a nickase Cas12J
protein, a dCas12J protein, a fusion Cas12J protein, etc.) and (b)
a Cas12J guide RNA (and/or a nucleic acid encoding the Cas12J guide
RNA). The present disclosure provides a nucleic acid/protein
complex (RNP complex) comprising: (a) a Cas12J polypeptide of the
present disclosure (e.g., where the Cas12J polypeptide can be a
naturally existing protein, a nickase Cas12J protein, a Cdas12J
protein, a fusion Cas12J protein, etc.); and (b) a Cas12J guide
RNA.
Cas12J Protein
[0102] A Cas12J polypeptide (this term is used interchangeably with
the term "Cas12J protein", "Cas.PHI. polypeptide", and "Cas.PHI.
protein") can bind and/or modify (e.g., cleave, nick, methylate,
demethylate, etc.) a target nucleic acid and/or a polypeptide
associated with target nucleic acid (e.g., methylation or
acetylation of a histone tail) (e.g., in some cases, the Cas12J
protein includes a fusion partner with an activity, and in some
cases, the Cas12J protein provides nuclease activity). In some
cases, the Cas12J protein is a naturally-occurring protein (e.g.,
naturally occurs in bacteriophage). In other cases, the Cas12J
protein is not a naturally-occurring polypeptide (e.g., the Cas12J
protein is a variant Cas12J protein (e.g., a catalytically inactive
Cas12J protein, a fusion Cas12J protein, and the like).
[0103] A Cas12J polypeptide (e.g., not fused to any heterologous
fusion partner) can have a molecular weight of from about 65
kiloDaltons (kDa) to about 85 kDa. For example, a Cas12J
polypeptide can have a molecular weight of from about 65 kDa to
about 70 kDa, from about 70 kDa to about 75 kDa, or from about 75
kDa to about 80 kDa. For example, a Cas12J polypeptide can have a
molecular weight of from about 70 kDa to about 80 kDa.
[0104] Assays to determine whether given protein interacts with a
Cas12J guide RNA can be any convenient binding assay that tests for
binding between a protein and a nucleic acid. Suitable binding
assays (e.g., gel shift assays) will be known to one of ordinary
skill in the art (e.g., assays that include adding a Cas12J guide
RNA and a protein to a target nucleic acid). Assays to determine
whether a protein has an activity (e.g., to determine if the
protein has nuclease activity that cleaves a target nucleic acid
and/or some heterologous activity) can be any convenient assay
(e.g., any convenient nucleic acid cleavage assay that tests for
nucleic acid cleavage). Suitable assays (e.g., cleavage assays)
will be known to one of ordinary skill in the art.
[0105] A naturally occurring Cas12J protein functions as an
endonuclease that catalyzes a double strand break at a specific
sequence in a targeted double stranded DNA (dsDNA). The sequence
specificity is provided by the associated guide RNA, which
hybridizes to a target sequence within the target DNA. The
naturally occurring Cas12J guide RNA is a crRNA, where the crRNA
includes (i) a guide sequence that hybridizes to a target sequence
in the target DNA and (ii) a protein binding segment which includes
a stem-loop (hairpin-dsRNA duplex) that binds to the Cas12J
protein.
[0106] In some cases, a C12J polypeptide of the present disclosure,
when complexed with a Cas12J guide RNA, generates a product nucleic
acid comprising 5' overhang following site specific cleavage of a
target nucleic acid. The 5' overhang can be an 8 to 12 nucleotide
(nt) overhang. For example, the 5' overhang can be 8 nt, 9 nt, 10
nt, 11, nt, or 12 nt in length.
[0107] In some embodiments, the Cas12J protein of the subject
methods and/or compositions is (or is derived from) a naturally
occurring (wild type) protein. Examples of naturally occurring
Cas12J proteins are depicted in FIG. 6A-6R. In some cases, a Cas12J
protein (of the subject compositions and/or methods) includes an
amino acid sequence having 20% or more sequence identity (e.g., 30%
or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or
more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or
more, 99% or more, or 100% sequence identity) with any one of the
Cas12J amino acid sequences depicted in FIG. 6 (e.g., any one of
FIG. 6A-6R). In some cases, a Cas12J protein (of the subject
compositions and/or methods) includes an amino acid sequence
depicted in FIG. 6 (e.g., any one of FIG. 6A-6R).
[0108] In some cases, a Cas12J protein (of the subject compositions
and/or methods) has more sequence identity to an amino acid
sequence depicted in FIG. 6 (e.g., any of the Cas12J amino acid
sequences depicted in FIG. 6) than to any of the following: Cas12a
proteins, Cas12b proteins, Cas12c proteins, Cas12d proteins, Cas12e
proteins, Cas12 g proteins, Cas12h proteins, and Cas12i proteins.
In some cases, a Cas12J protein (of the subject compositions and/or
methods) includes an amino acid sequence having a RuvC domain
(which includes the RuvC-I, RuvC-II, and RuvC-III domains) that has
more sequence identity to the RuvC domain of an amino acid sequence
depicted in FIG. 6 (e.g., the RuvC domain of any of the Cas12J
amino acid sequences depicted in FIG. 6) than to the RuvC domain of
any of the following: Cas12a proteins, Cas12b proteins, Cas12c
proteins, Cas12d proteins, Cas12e proteins, Cas12 g proteins,
Cas12h proteins, and Cas12i proteins.
[0109] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the RuvC domain (which includes the RuvC-I, RuvC-II,
and RuvC-III domains) of any one of the Cas12J amino acid sequences
depicted in FIG. 6 (e.g., any one of FIG. 6A-6R). In some cases, a
Cas12J protein (of the subject compositions and/or methods)
includes an amino acid sequence having 70% or more sequence
identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more,
95% or more, 97% or more, 98% or more, 99% or more, or 100%
sequence identity) with the RuvC domain (which includes the RuvC-I,
RuvC-II, and RuvC-III domains) of any one of the Cas12J amino acid
sequences depicted in FIG. 6 (e.g., any one of FIG. 6A-6R). In some
cases, a Cas12J protein (of the subject compositions and/or
methods) includes the RuvC domain (which includes the RuvC-I,
RuvC-II, and RuvC-III domains) of any one of the Cas12J amino acid
sequences depicted in FIG. 6 (e.g., any one of FIG. 6A-6R).
[0110] In some cases, a guide RNA that binds a Cas12J polypeptide
includes a nucleotide sequence depicted in FIG. 7 (or in some cases
the reverse complement of same). In some cases, the guide RNA
comprises the nucleotide sequence (N)nX or the reverse complement
of same, where N is any nucleotide, n is an integer from 15 to 30
(e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22,
from 18 to 20, from 20 to 25, or from 25 to 30), and X is any one
of the nucleotide sequences depicted in FIG. 7 (or in some cases
the reverse complement of same).
[0111] In some cases, a guide RNA that binds a Cas12J polypeptide
includes a nucleotide sequence having 20% or more sequence identity
(e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100% sequence identity) with any
one of the sequences depicted in FIG. 7 (or in some cases the
reverse complement of same). In some cases, the guide RNA comprises
the nucleotide sequence (N)nX or the reverse complement of same,
where N is any nucleotide, n is an integer from 15 to 30 (e.g.,
from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18
to 20, from 20 to 25, or from 25 to 30), and X a nucleotide
sequence having 20% or more sequence identity (e.g., 30% or more,
40% or more, 50% or more, 60% or more, 70% or more, 80% or more,
85% or more, 90% or more, 95% or more, 97% or more, 98% or more,
99% or more, or 100% sequence identity) with any one of the
sequences depicted in FIG. 7.
[0112] In some cases, a guide RNA that binds a Cas12J polypeptide
includes a nucleotide sequence having 85% or more sequence identity
(e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with any one of the sequences
depicted in FIG. 7 (or in some cases the reverse complement of
same). In some cases, the guide RNA comprises the nucleotide
sequence (N)nX or the reverse complement of same, where N is any
nucleotide, n is an integer from 15 to 30 (e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30), and X a nucleotide sequence having 85% or
more sequence identity (e.g., 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100% sequence identity) with any
one of the sequences depicted in FIG. 7.
[0113] In some cases, a guide RNA that binds a Cas12J polypeptide
includes a nucleotide sequence depicted in FIG. 7 (or in some cases
the reverse complement of same). In some cases, the guide RNA
comprises the nucleotide sequence X(N)n, where N is any nucleotide,
n is an integer from 15 to 30 (e.g., from 15 to 20, from 17 to 25,
from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from
25 to 30), and X is any one of the nucleotide sequences depicted in
FIG. 7 (or in some cases the reverse complement of same).
[0114] In some cases, a guide RNA that binds a Cas12J polypeptide
includes a nucleotide sequence having 20% or more sequence identity
(e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100% sequence identity) with any
one of the sequences depicted in FIG. 7 (or in some cases the
reverse complement of same). In some cases, the guide RNA comprises
the nucleotide sequence X(N)n, where N is any nucleotide, n is an
integer from 15 to 30 (e.g., from 15 to 20, from 17 to 25, from 17
to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to
30), and X a nucleotide sequence having 20% or more sequence
identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more,
70% or more, 80% or more, 85% or more, 90% or more, 95% or more,
97% or more, 98% or more, 99% or more, or 100% sequence identity)
with any one of the sequences depicted in FIG. 7.
[0115] Examples of Cas12J proteins are depicted in FIG. 6A-6R. As
noted above, a Cas12J polypeptide is also referred to herein as a
"Cas.PHI. polypeptide." For example:
[0116] 1) the Cas12J polypeptide designated "Cas12J_1947455" (or
"Cas12J_1947455_11" in FIG. 9) and depicted in FIG. 6A is also
referred to herein as "Cas.PHI.-1";
[0117] 2) the Cas12J polypeptide designated "Cas12J_2071242" and
depicted in FIG. 6B is also referred to herein as "Cas.PHI.-2"
[0118] 3) the Cas12J polypeptide designated "Cas12J_3339380 (or
"Cas12J_3339380_12" in FIG. 9) and depicted in FIG. 6D is also
referred to herein as "Cas.PHI.-3";
[0119] 4) the Cas12J polypeptide designated "Cas12J_3877103_16" and
depicted in FIG. 6Q is also referred to herein as "Cas.PHI.-4";
[0120] 5) the Cas12J polypeptide designated "Cas12J_10000002_47" or
"Cas12J_1000002_112" and depicted in FIG. 6G is also referred to
herein as "Cas.PHI.-5";
[0121] 6) the Cas12J polypeptide designated "Cas12J_10100763_4" and
depicted in FIG. 6H is also referred to herein as "Cas.PHI.-6";
[0122] 7) the Cas12J polypeptide designated "Cas12J_1000007_143" or
"Cas12J_1000001_267" and depicted in FIG. 6P is also referred to
herein as "Cas.PHI.-7";
[0123] 8) the Cas12J polypeptide designated "Cas12J_10000286_53"
and depicted in FIG. 6L (or "Cas12J_10000506_8" and depicted in
FIG. 6O) is also referred to herein as "Cas.PHI.-8";
[0124] 9) the Cas12J polypeptide designated "Cas12J_10001283_7" and
depicted in FIG. 6M is also referred to herein as "Cas.PHI.-9";
[0125] 10) the Cas12J polypeptide designated "Cas12J_10037042_3"
and depicted in FIG. 6E is also referred to herein as
"Cas.PHI.-10".
[0126] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6A
and designated "Cas12J_1947455." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6A. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6A. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6A. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6A. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6A, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 680
amino acids (aa) to 720 aa, e.g., from 680 aa to 690 aa, from 690
aa to 700 aa, from 700 aa to 710 aa, or from 710 aa to 720 aa). In
some cases, the Cas12J polypeptide has a length of 707 amino acids.
In some cases, a guide RNA that binds a Cas12J polypeptide (e.g., a
Cas12J polypeptide comprising an amino acid sequence having 20% or
more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100%, amino acid sequence
identity to the Cas12J amino acid sequence depicted in FIG. 6A.)
includes the following nucleotide sequence:
GTCTCGACTAATCGAGCAATCGTTTGAGATCTCTCC (SEQ ID NO: 1) or the reverse
complement of same. In some cases, the guide RNA comprises the
nucleotide sequence (N)nGTCTCGACTAATCGAGCAATCGTTTGAGATCTCTCC (SEQ
ID NO: 2) or the reverse complement of same, where N is any
nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30). The Cas12J protein designated
Cas12J_1947455 (or Cas12J_1947455_11 in FIG. 9), and depicted in
FIG. 6A, is also referred to herein as "ortholog #1" or
"Cas12.PHI.-1."
[0127] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6B
and designated "Cas12J_071242." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6B. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6B. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6B. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6B. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6B, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 740
amino acids (aa) to 780 aa, e.g., from 740 aa to 750 aa, from 750
aa to 760 aa, from 760 aa to 770 aa, or from 770 aa to 780 aa). In
some cases, the Cas12J polypeptide has a length of 757 amino acids.
In some cases, a guide RNA that binds a Cas12J polypeptide (e.g., a
Cas12J polypeptide comprising an amino acid sequence having 20% or
more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100%, amino acid sequence
identity to the Cas12J amino acid sequence depicted in FIG. 6B)
includes the following nucleotide sequence:
GTCGGAACGCTCAACGATTGCCCCTCACGAGGGGAC (SEQ ID NO: 3) or the reverse
complement of same. In some cases, the guide RNA comprises the
nucleotide sequence (N)nGTCGGAACGCTCAACGATTGCCCCTCACGAGGGGAC (SEQ
ID NO: 4) or the reverse complement of same, where N is any
nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30). The Cas12J protein designated
Cas12J_2071242, and depicted in FIG. 6B, is also referred to herein
as "ortholog #2" or "Cas12.PHI.-2."
[0128] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6C
and designated "Cas12J_1973640." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6C. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6C. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6C. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6C. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6C, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 740
amino acids (aa) to 780 aa, e.g., from 740 aa to 750 aa, from 750
aa to 760 aa, from 760 aa to 770 aa, or from 770 aa to 780 aa). In
some cases, the Cas12J polypeptide has a length of 765 amino
acids.
[0129] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6D
and designated "Cas12J_3339380." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6D. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6D. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6D. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6D. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6D, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 740
amino acids (aa) to 780 aa, e.g., from 740 aa to 750 aa, from 750
aa to 760 aa, from 760 aa to 770 aa, or from 770 aa to 780 aa). In
some cases, the Cas12J polypeptide has a length of 766 amino acids.
In some cases, a guide RNA that binds a Cas12J polypeptide (e.g., a
Cas12J polypeptide comprising an amino acid sequence having 20% or
more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100%, amino acid sequence
identity to the Cas12J amino acid sequence depicted in FIG. 6D)
includes the following nucleotide sequence:
GTCCCAGCGTACTGGGCAATCAATAGTCGTTTTGGT (SEQ ID NO: 5) or the reverse
complement of same. In some cases, the guide RNA comprises the
nucleotide sequence (N)nGTCCCAGCGTACTGGGCAATCAATAGTCGTTTTGGT (SEQ
ID NO: 6) or the reverse complement of same, where N is any
nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30). The Cas12J protein designated
Cas12J_3339380, and depicted in FIG. 6D, is also referred to herein
as "ortholog #3" or "Cas12.PHI.-3."
[0130] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6E
and designated "Cas12J_10037042_3." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6E. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6E. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6E. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6E. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6E, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 780
amino acids (aa) to 820 aa, e.g., from 780 aa to 790 aa, from 790
aa to 800 aa, from 800 aa to 810 aa, or from 810 aa to 820 aa). In
some cases, the Cas12J polypeptide has a length of 812 amino
acids.
[0131] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6F
and designated "Cas12J_10020921_9." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6F. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6F. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6F. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6F. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6F, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 780
amino acids (aa) to 820 aa, e.g., from 780 aa to 790 aa, from 790
aa to 800 aa, from 800 aa to 810 aa, or from 810 aa to 820 aa). In
some cases, the Cas12J polypeptide has a length of 812 amino
acids.
[0132] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6G
and designated "Cas12J_10000002_47." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6G. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6G. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6G. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6G. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6G, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 770
amino acids (aa) to 810 aa, e.g., from 770 aa to 780 aa, from 780
aa to 790 aa, from 790 aa to 800 aa, or from 800 aa to 810 aa). In
some cases, the Cas12J polypeptide has a length of 793 amino acids.
In some cases, a guide RNA that binds a Cas12J polypeptide (e.g., a
Cas12J polypeptide comprising an amino acid sequence having 20% or
more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100%, amino acid sequence
identity to the Cas12J amino acid sequence depicted in FIG. 6G)
includes the following nucleotide sequence:
GGATCCAATCCTTTTTGATTGCCCAATTCGTTGGGAC (SEQ ID NO: 7) or the reverse
complement of same. In some cases, the guide RNA comprises the
nucleotide sequence (N)nGGATCCAATCCTTTTTGATTGCCCAATTCGTTGGGAC (SEQ
ID NO: 8) or the reverse complement of same, where N is any
nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30.
[0133] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6H
and designated "Cas12J_10100763_4." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6H. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6H. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6H. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6H. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6H, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 420
amino acids (aa) to 460 aa, e.g., from 420 aa to 430 aa, from 430
aa to 440 aa, from 440 aa to 450 aa, or from 450 aa to 460 aa). In
some cases, the Cas12J polypeptide has a length of 441 amino
acids.
[0134] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6I
and designated "Cas12J_10004149_10." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6I. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6I. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6I. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6I. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6I, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 790
amino acids (aa) to 830 aa, e.g., from 790 aa to 800 aa, from 800
aa to 810 aa, from 810 aa to 820 aa, or rom 820 aa to 830 aa). In
some cases, the Cas12J polypeptide has a length of 812 amino
acids.
[0135] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6J
and designated "Cas12J_10000724_71." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6J. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6J. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6J. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6J. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6J, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 790
amino acids (aa) to 830 aa, e.g., from 790 aa to 800 aa, from 800
aa to 810 aa, from 810 aa to 820 aa, or from 820 aa to 830 aa). In
some cases, the Cas12J polypeptide has a length of 812 amino acids.
In some cases, a guide RNA that binds a Cas12J polypeptide (e.g., a
Cas12J polypeptide comprising an amino acid sequence having 20% or
more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100%, amino acid sequence
identity to the Cas12J amino acid sequence depicted in FIG. 6J)
includes the following nucleotide sequence:
GGATCTGAGGATCATTATTGCTCGTTACGACGAGAC (SEQ ID NO: 9) or the reverse
complement of same. In some cases, the guide RNA comprises the
nucleotide sequence (N)nGGATCTGAGGATCATTATTGCTCGTTACGACGAGAC (SEQ
ID NO: 10) or the reverse complement of same, where N is any
nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30. In some cases, a guide RNA that binds a
Cas12J polypeptide (e.g., a Cas12J polypeptide comprising an amino
acid sequence having 20% or more, 30% or more, 40% or more, 50% or
more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or
more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%,
amino acid sequence identity to the Cas12J amino acid sequence
depicted in FIG. 6J) includes the following nucleotide sequence:
GTCTCGTCGTAACGAGCAATAATGATCCTCAGATCC (SEQ ID NO: 11) or the reverse
complement of same. In some cases, the guide RNA comprises the
nucleotide sequence (N)n GTCTCGTCGTAACGAGCAATAATGATCCTCAGATCC (SEQ
ID NO: 12) or the reverse complement of same, where N is any
nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30.
[0136] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6K
and designated "Cas12J_1000001_267." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6K. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6K. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6K. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6K. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6K, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 750
amino acids (aa) to 790 aa, e.g., from 750 aa to 760 aa, from 760
aa to 770 aa, from 770 aa to 780 aa, or from 780 aa to 790 aa). In
some cases, the Cas12J polypeptide has a length of 772 amino acids.
In some cases, a guide RNA that binds a Cas12J polypeptide (e.g., a
Cas12J polypeptide comprising an amino acid sequence having 20% or
more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100%, amino acid sequence
identity to the Cas12J amino acid sequence depicted in FIG. 6K)
includes the following nucleotide sequence:
GTCTCAGCGTACTGAGCAATCAAAAGGTTTCGCAGG (SEQ ID NO: 13) or the reverse
complement of same. In some cases, the guide RNA comprises the
nucleotide sequence (N)nGTCTCAGCGTACTGAGCAATCAAAAGGTTTCGCAGG (SEQ
ID NO: 14) or the reverse complement of same, where N is any
nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30.
[0137] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6L
and designated "Cas12J_10000286_53." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6L. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6L. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6L. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6L. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6L, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 700
amino acids (aa) to 740 aa, e.g., from 700 aa to 710 aa, from 710
aa to 720 aa, from 720 aa to 730 aa, or from 730 aa to 740 aa). In
some cases, the Cas12J polypeptide has a length of 717 amino acids.
In some cases, a guide RNA that binds a Cas12J polypeptide (e.g., a
Cas12J polypeptide comprising an amino acid sequence having 20% or
more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100%, amino acid sequence
identity to the Cas12J amino acid sequence depicted in FIG. 6L)
includes the following nucleotide sequence:
GTCTCCTCGTAAGGAGCAATCTATTAGTCTTGAAAG (SEQ ID NO: 15) or the reverse
complement of same. In some cases, the guide RNA comprises the
nucleotide sequence (N)nGTCTCCTCGTAAGGAGCAATCTATTAGTCTTGAAAG (SEQ
ID NO: 16) or the reverse complement of same, where N is any
nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30.
[0138] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6M
and designated "Cas12J_10001283_7." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6M. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6M. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6M. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6M. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6M, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 770
amino acids (aa) to 810 aa, e.g., from 770 aa to 780 aa, from 780
aa to 790 aa, from 790 aa to 800 aa, or from 800 aa to 810 aa). In
some cases, the Cas12J polypeptide has a length of 793 amino acids.
In some cases, a guide RNA that binds a Cas12J polypeptide (e.g., a
Cas12J polypeptide comprising an amino acid sequence having 20% or
more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100%, amino acid sequence
identity to the Cas12J amino acid sequence depicted in FIG. 6M)
includes the following nucleotide sequence:
GTCTCGGCGCACCGAGCAATCAGCGAGGTCTTCTAC (SEQ ID NO: 17) or the reverse
complement of same. In some cases, the guide RNA comprises the
nucleotide sequence (N)nGTCTCGGCGCACCGAGCAATCAGCGAGGTCTTCTAC (SEQ
ID NO: 18) or the reverse complement of same, where N is any
nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30.
[0139] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6N
and designated "Cas12J_1000002_112." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6N. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6N. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6N. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6N. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6N, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 770
amino acids (aa) to 810 aa, e.g., from 770 aa to 780 aa, from 780
aa to 790 aa, from 790 aa to 800 aa, or from 800 aa to 810 aa). In
some cases, the Cas12J polypeptide has a length of 793 amino acids.
In some cases, a guide RNA that binds a Cas12J polypeptide (e.g., a
Cas12J polypeptide comprising an amino acid sequence having 20% or
more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100%, amino acid sequence
identity to the Cas12J amino acid sequence depicted in FIG. 6N)
includes the following nucleotide sequence:
GTCCCAACGAATTGGGCAATCAAAAAGGATTGGATCC (SEQ ID NO: 19) or the
reverse complement of same. In some cases, the guide RNA comprises
the nucleotide sequence (N)nGTCCCAACGAATTGGGCAATCAAAAAGGATTGGATCC
(SEQ ID NO: 20) or the reverse complement of same, where N is any
nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30.
[0140] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6O
and designated "Cas12J_10000506_8." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6O. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6O. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6O. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6O. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6O, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 700
amino acids (aa) to 740 aa, e.g., from 700 aa to 710 aa, from 710
an to 720 aa, from 720 aa to 730 aa, or from 730 aa to 740 aa). In
some cases, the Cas12J polypeptide has a length of 717 amino acids.
In some cases, a guide RNA that binds a Cas12J polypeptide (e.g., a
Cas12J polypeptide comprising an amino acid sequence having 20% or
more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100%, amino acid sequence
identity to the Cas12J amino acid sequence depicted in FIG. 6O)
includes the following nucleotide sequence:
GTCTCCTCGTAAGGAGCAATCTATTAGTCTTGAAAG (SEQ ID NO: 15) or the reverse
complement of same. In some cases, the guide RNA comprises the
nucleotide sequence (N)nGTCTCCTCGTAAGGAGCAATCTATTAGTCTTGAAAG (SEQ
ID NO: 16) or the reverse complement of same, where N is any
nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30.
[0141] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6P
and designated "Cas12J_1000007_143." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6P. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6P. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6P. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6P. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6P, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 750
amino acids (aa) to 790 aa, e.g., from 750 aa to 760 aa, from 760
aa to 770 aa, from 770 aa to 780 aa, or from 780 aa to 790 aa). In
some cases, the Cas12J polypeptide has a length of 772 amino acids.
In some cases, a guide RNA that binds a Cas12J polypeptide (e.g., a
Cas12J polypeptide comprising an amino acid sequence having 20% or
more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100%, amino acid sequence
identity to the Cas12J amino acid sequence depicted in FIG. 6P)
includes the following nucleotide sequence:
GTCTCAGCGTACTGAGCAATCAAAAGGTTTCGCAGG (SEQ ID NO: 13) or the reverse
complement of same. In some cases, the guide RNA comprises the
nucleotide sequence (N)nGTCTCAGCGTACTGAGCAATCAAAAGGTTTCGCAGG (SEQ
ID NO: 14) or the reverse complement of same, where N is any
nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30.
[0142] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6Q
and designated "Cas12J_3877103_16." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6Q. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6Q. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6Q. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6Q. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6Q, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 750
amino acids (aa) to 790 aa, e.g., from 750 aa to 760 aa, from 760
aa to 770 aa, from 770 aa to 780 aa, or from 780 aa to 790 aa). In
some cases, the Cas12J polypeptide has a length of 765 amino acids.
In some cases, a guide RNA that binds a Cas12J polypeptide (e.g., a
Cas12J polypeptide comprising an amino acid sequence having 20% or
more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100%, amino acid sequence
identity to the Cas12J amino acid sequence depicted in FIG. 6Q)
includes the following nucleotide sequence:
GTCGCGGCGTACCGCGCAATGAGAGTCTGTTGCCAT (SEQ ID NO: 21) or the reverse
complement of same. In some cases, the guide RNA comprises the
nucleotide sequence (N)n GTCGCGGCGTACCGCGCAATGAGAGTCTGTTGCCAT (SEQ
ID NO: 22) or the reverse complement of same, where N is any
nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30.
[0143] In some cases, a Cas12J protein (of the subject compositions
and/or methods) includes an amino acid sequence having 20% or more
sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60%
or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100% sequence
identity) with the Cas12J amino acid sequence depicted in FIG. 6R
and designated "Cas12J_877636_12." For example, in some cases, a
Cas12J protein includes an amino acid sequence having 50% or more
sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6R. In some cases, a Cas12J protein
includes an amino acid sequence having 80% or more sequence
identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more,
98% or more, 99% or more, or 100% sequence identity) with the
Cas12J amino acid sequence depicted in FIG. 6R. In some cases, a
Cas12J protein includes an amino acid sequence having 90% or more
sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99%
or more, or 100% sequence identity) with the Cas12J amino acid
sequence depicted in FIG. 6R. In some cases, a Cas12J protein
includes an amino acid sequence having the Cas12J protein sequence
depicted in FIG. 6R. In some cases, a Cas12J protein includes an
amino acid sequence having the Cas12J protein sequence depicted in
FIG. 6R, with the exception that the sequence includes an amino
acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that
reduces the naturally occurring catalytic activity of the protein.
In some cases, the Cas12J polypeptide has a length of from 750
amino acids (aa) to 790 aa, e.g., from 750 aa to 760 aa, from 760
aa to 770 aa, from 770 aa to 780 aa, or from 780 aa to 790 aa). In
some cases, the Cas12J polypeptide has a length of 766 amino acids.
In some cases, a guide RNA that binds a Cas12J polypeptide (e.g., a
Cas12J polypeptide comprising an amino acid sequence having 20% or
more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or
more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100%, amino acid sequence
identity to the Cas12J amino acid sequence depicted in FIG. 6R)
includes the following nucleotide sequence:
ACCAAAACGACTATTGATTGCCCAGTACGCTGGGAC (SEQ ID NO: 23) or the reverse
complement of same. In some cases, the guide RNA comprises the
nucleotide sequence (N)n ACCAAAACGACTATTGATTGCCCAGTACGCTGGGAC (SEQ
ID NO: 24) or the reverse complement of same, where N is any
nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20,
from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20
to 25, or from 25 to 30.
Cas12J Variants
[0144] A variant Cas12J protein has an amino acid sequence that is
different by at least one amino acid (e.g., has a deletion,
insertion, substitution, fusion) when compared to the amino acid
sequence of the corresponding wild type Cas12J protein, e.g., when
compared to the Cas12J amino acid sequence depicted in any one of
FIG. 6A-6R. In some cases, a Cas12J variant comprises from 1 amino
acid substitution to 10 amino acid substitutions compared to the
Cas12J amino acid sequence depicted in any one of FIG. 6A-6R. In
some cases, a Cas12J variant comprises from 1 amino acid
substitution to 10 amino acid substitutions in the RuvC domain,
compared to the Cas12J amino acid sequence depicted in any one of
FIG. 6A-6R.
Variants--Catalytic Activity
[0145] In some cases, the Cas12J protein is a variant Cas12J
protein, e.g., mutated relative to the naturally occurring
catalytically active sequence, and exhibits reduced cleavage
activity (e.g., exhibits 90%, or less, 80% or less, 70% or less,
60% or less, 50% or less, 40% or less, or 30% or less cleavage
activity) when compared to the corresponding naturally occurring
sequence. In some cases, such a variant Cas12J protein is a
catalytically `dead` protein (has substantially no cleavage
activity) and can be referred to as a `dCas12J.` In some cases, the
variant Cas12J protein is a nickase (cleaves only one strand of a
double stranded target nucleic acid, e.g., a double stranded target
DNA). As described in more detail herein, in some cases, a Cas12J
protein (in some case a Cas12J protein with wild type cleavage
activity and in some cases a variant Cas12J with reduced cleavage
activity, e.g., a dCas12J or a nickase Cas12J) is fused
(conjugated) to a heterologous polypeptide that has an activity of
interest (e.g., a catalytic activity of interest) to form a fusion
protein (a fusion Cas12J protein).
[0146] Amino acid substitutions that result in a Cas12J polypeptide
that, when complexed with a Cas12J guide RNA, binds, but does not
cleave, a target nucleic acid are depicted in FIG. 9. For example,
a substitution of the Asp at position 464 of Cas12J_10037042_3, or
a corresponding position in another Cas12J, results in a dCas12J.
As another example, a substitution of the Glu at position 678 of
Cas12J_10037042_3, or a corresponding position in another Cas12J,
results in a dCas12J. As another example, a substation of the Asp
at position 769 of Cas12J_10037042_3, or a corresponding position
in another Cas12J, results in a dCas12J.
[0147] An amino acid substitution that results in a dCas12J
polypeptide (i.e., a Cas12J polypeptide that binds, but does not
cleave, a target nucleic acid when complexed with a guide RNA)
includes a substitution of the Asp at position 413 of
Cas12J_3339380 (FIG. 6D), or a corresponding position in another
Cas12J, with an amino acid other than Asp. As an example, an amino
acid substitution that results in a dCas12J polypeptide (i.e., a
Cas12J polypeptide that binds, but does not cleave, a target
nucleic acid when complexed with a guide RNA) includes a D413A
substitution at position 413 of Cas12J_3339380 (FIG. 6D), or a
corresponding position in another Cas12J.
[0148] An amino acid substitution that results in a dCas12J
polypeptide (i.e., a Cas12J polypeptide that binds, but does not
cleave, a target nucleic acid when complexed with a guide RNA)
includes a substitution of the Asp at position 371 of
Cas12J_1947455 (FIG. 6A), or a corresponding position in another
Cas12J, with an amino acid other than Asp. As an example, an amino
acid substitution that results in a dCas12J polypeptide (i.e., a
Cas12J polypeptide that binds, but does not cleave, a target
nucleic acid when complexed with a guide RNA) includes a D371A
substitution at position 371 of Cas12J_1947455 (FIG. 6A), or a
corresponding position in another Cas12J.
[0149] An amino acid substitution that results in a dCas12J
polypeptide (i.e., a Cas12J polypeptide that binds, but does not
cleave, a target nucleic acid when complexed with a guide RNA)
includes a substitution of the Asp at position 394 of
Cas12J_2071242 (FIG. 6B), or a corresponding position in another
Cas12J, with an amino acid other than Asp. As an example, an amino
acid substitution that results in a dCas12J polypeptide (i.e., a
Cas12J polypeptide that binds, but does not cleave, a target
nucleic acid when complexed with a guide RNA) includes a D394A
substitution at position 394 of Cas12J_2071242 (FIG. 6B), or a
corresponding position in another Cas12J.
[0150] Amino acid positions corresponding to the Asp at position
413 of Cas12J_3339380 (FIG. 6D) (Cas.PHI.-3), the Asp at position
371 of Cas12J_1947455 (FIG. 6A) (Cas.PHI.-1), and the Asp at
position 394 of Cas12J_2071242 (FIG. 6B) (Cas.PHI.-2), can be
readily determined by, e.g., aligning the amino acid sequences of
the Cas12J polypeptides depicted in FIG. 6A-6R. For example, amino
acid positions corresponding to the Asp at position 413 of
Cas12J_3339380 (FIG. 6D), the Asp at position 371 of Cas12J_1947455
(FIG. 6A), and the Asp at position 394 of Cas12J_2071242 (FIG. 6B),
are depicted in FIG. 9. For example, the Asp in Ruv-CI that, when
substituted with an amino acid other than Asp, can in a dCas12J
polypeptide includes:
[0151] 1) Asp-371 of the Cas12J polypeptide designated
"Cas12J_1947455" (or "Cas12J_1947455_11" in FIG. 9) and depicted in
FIG. 6A ("Cas.PHI.-1");
[0152] 2) Asp-394 of the Cas12J polypeptide designated
"Cas12J_2071242" and depicted in FIG. 6B ("Cas.PHI.-2");
[0153] 3) Asp-413 of the Cas12J polypeptide designated
"Cas12J_3339380 (or "Cas12J_3339380_12" in FIG. 9) and depicted in
FIG. 6D ("Cas.PHI.-3");
[0154] 4) Asp-419 of the Cas12J polypeptide designated
"Cas12J_3877103_16" and depicted in FIG. 6Q ("Cas.PHI.-4");
[0155] 5) Asp-416 of the Cas12J polypeptide designated
"Cas12J_10000002_47" or "Cas12J_1000002_112" and depicted in FIG.
6G ("Cas.PHI.-5");
[0156] 6) Asp-384 of the Cas12J polypeptide designated
"Cas12J_10100763_4" and depicted in FIG. 6H ("Cas.PHI.-6");
[0157] 7) Asp-423 of the Cas12J polypeptide designated
"Cas12J_1000007_143" or "Cas12J_1000001_267" and depicted in FIG.
6P ("Cas.PHI.-7");
[0158] 8) Asp-369 of the Cas12J polypeptide designated
"Cas12J_10000286_53" and depicted in FIG. 6L (or
"Cas12J_10000506_8" and depicted in FIG. 6O) ("Cas.PHI.-8");
[0159] 9) Asp-426 of the Cas12J polypeptide designated
"Cas12J_10001283_7" and depicted in FIG. 6M ("Cas.PHI.-9");
[0160] 10) Asp-464 of the Cas12J polypeptide designated
"Cas12J_10037042_3" and depicted in FIG. 6E ("Cas.PHI.-10").
Variants--Fusion Cas12J Polypeptides
[0161] As noted above, in some cases, a Cas12J protein (in some
cases a Cas12J protein with wild type cleavage activity and in some
cases a variant Cas12J with reduced cleavage activity, e.g., a
dCas12J or a nickase Cas12J) is fused (conjugated) to a
heterologous polypeptide (i.e., one or more heterologous
polypeptides) that has an activity of interest (e.g., a catalytic
activity of interest) to form a fusion protein. A heterologous
polypeptide to which a Cas12J protein can be fused is referred to
herein as a "fusion partner."
[0162] In some cases, the fusion partner can modulate transcription
(e.g., inhibit transcription, increase transcription) of a target
DNA. For example, in some cases the fusion partner is a protein (or
a domain from a protein) that inhibits transcription (e.g., a
transcriptional repressor, a protein that functions via recruitment
of transcription inhibitor proteins, modification of target DNA
such as methylation, recruitment of a DNA modifier, modulation of
histones associated with target DNA, recruitment of a histone
modifier such as those that modify acetylation and/or methylation
of histones, and the like). In some cases, the fusion partner is a
protein (or a domain from a protein) that increases transcription
(e.g., a transcription activator, a protein that acts via
recruitment of transcription activator proteins, modification of
target DNA such as demethylation, recruitment of a DNA modifier,
modulation of histones associated with target DNA, recruitment of a
histone modifier such as those that modify acetylation and/or
methylation of histones, and the like). In some cases, the fusion
partner is a reverse transcriptase. In some cases, the fusion
partner is a base editor. In some cases, the fusion partner is a
deaminase.
[0163] In some cases, a fusion Cas12J protein includes a
heterologous polypeptide that has enzymatic activity that modifies
a target nucleic acid (e.g., nuclease activity, methyltransferase
activity, demethylase activity, DNA repair activity, DNA damage
activity, deamination activity, dismutase activity, alkylation
activity, depurination activity, oxidation activity, pyrimidine
dimer forming activity, integrase activity, transposase activity,
recombinase activity, polymerase activity, ligase activity,
helicase activity, photolyase activity, or glycosylase
activity).
[0164] In some cases, a fusion Cas12J protein includes a
heterologous polypeptide that has enzymatic activity that modifies
a polypeptide (e.g., a histone) associated with a target nucleic
acid (e.g., methyltransferase activity, demethylase activity,
acetyltransferase activity, deacetylase activity, kinase activity,
phosphatase activity, ubiquitin ligase activity, deubiquitinating
activity, adenylation activity, deadenylation activity, SUMOylating
activity, deSUMOylating activity, ribosylation activity,
deribosylation activity, myristoylation activity or
demyristoylation activity).
[0165] Examples of proteins (or fragments thereof) that can be used
in increase transcription include but are not limited to:
transcriptional activators such as VP16, VP64, VP48, VP160, p65
subdomain (e.g., from NFkB), and activation domain of EDLL and/or
TAL activation domain (e.g., for activity in plants); histone
lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1,
SYMD2, NSD1, and the like; histone lysine demethylases such as
JHDM2a/b, UTX, JMJD3, and the like; histone acetyltransferases such
as GCN5, PCAF, CBP, p300, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4,
SRC1, ACTR, P160, CLOCK, and the like; and DNA demethylases such as
Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME,
DML1, DML2, ROS1, and the like.
[0166] Examples of proteins (or fragments thereof) that can be used
in decrease transcription include but are not limited to:
transcriptional repressors such as the Kruppel associated box (KRAB
or SKD); KOX1 repression domain; the Mad mSIN3 interaction domain
(SID); the ERF repressor domain (ERD), the SRDX repression domain
(e.g., for repression in plants), and the like; histone lysine
methyltransferases such as Pr-SET7/8, SUV4-20H1, RIZI, and the
like; histone lysine demethylases such as JMJD2A/JHDM3A, JMJD2B,
JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX,
JARID1D/SMCY, and the like; histone lysine deacetylases such as
HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1,
SIRT2, HDAC11, and the like; DNA methylases such as HhaI DNA
m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1),
DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b
(DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the
like; and periphery recruitment elements such as Lamin A, Lamin B,
and the like.
[0167] In some cases, the fusion partner has enzymatic activity
that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA,
dsDNA). Examples of enzymatic activity that can be provided by the
fusion partner include but are not limited to: nuclease activity
such as that provided by a restriction enzyme (e.g., FokI
nuclease), methyltransferase activity such as that provided by a
methyltransferase (e.g., HhaI DNA m5c-methyltransferase (M.HhaI),
DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a),
DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2,
CMT1, CMT2 (plants), and the like); demethylase activity such as
that provided by a demethylase (e.g., Ten-Eleven Translocation
(TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the
like), DNA repair activity, DNA damage activity, deamination
activity such as that provided by a deaminase (e.g., a cytosine
deaminase enzyme such as rat APOBEC1), dismutase activity,
alkylation activity, depurination activity, oxidation activity,
pyrimidine dimer forming activity, integrase activity such as that
provided by an integrase and/or resolvase (e.g., Gin invertase such
as the hyperactive mutant of the Gin invertase, GinH106Y; human
immunodeficiency virus type 1 integrase (IN); Tn3 resolvase; and
the like), transposase activity, recombinase activity such as that
provided by a recombinase (e.g., catalytic domain of Gin
recombinase), polymerase activity, ligase activity, helicase
activity, photolyase activity, and glycosylase activity).
[0168] In some cases, the fusion partner has enzymatic activity
that modifies a protein associated with the target nucleic acid
(e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNA binding
protein, a DNA binding protein, and the like). Examples of
enzymatic activity (that modifies a protein associated with a
target nucleic acid) that can be provided by the fusion partner
include but are not limited to: methyltransferase activity such as
that provided by a histone methyltransferase (HMT) (e.g.,
suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as
KMTIA), euchromatic histone lysine methyltransferase 2 (G9A, also
known as KMT1C and EHMT2), SUV39H2, ESET/SETDB1, and the like,
SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7/8,
SUV4-20H1, EZH2, RIZI), demethylase activity such as that provided
by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also
known as LSD1), JHDM2a/b, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1,
JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY,
UTX, JMJD3, and the like), acetyltransferase activity such as that
provided by a histone acetylase transferase (e.g., catalytic
core/fragment of the human acetyltransferase p300, GCN5, PCAF, CBP,
TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, HBO1/MYST2, HMOF/MYST1,
SRC1, ACTR, P160, CLOCK, and the like), deacetylase activity such
as that provided by a histone deacetylase (e.g., HDAC1, HDAC2,
HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and
the like), kinase activity, phosphatase activity, ubiquitin ligase
activity, deubiquitinating activity, adenylation activity,
deadenylation activity, SUMOylating activity, deSUMOylating
activity, ribosylation activity, deribosylation activity,
myristoylation activity, and demyristoylation activity.
[0169] Additional examples of a suitable fusion partners are
dihydrofolate reductase (DHFR) destabilization domain (e.g., to
generate a chemically controllable fusion Cas12J protein), and a
chloroplast transit peptide. Suitable chloroplast transit peptides
include, but are not limited to:
TABLE-US-00001 (SEQ ID NO: 25)
MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSIT
SNGGRVKCMQVWPPIGKKKFETLSYLPPLTRDSRA; (SEQ ID NO: 26)
MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSIT SNGGRVKS; (SEQ ID
NO: 27) MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSN
GGRVNCMQVWPPIEKKKFETLSYLPDLTDSGGRVNC; (SEQ ID NO: 28)
MAQVSRICNGVQNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSW
GLKKSGMTLIGSELRPLKVMSSVSTAC; (SEQ ID NO: 29)
MAQVSRICNGVWNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSW
GLKKSGMTLIGSELRPLKVMSSVSTAC; (SEQ ID NO: 30)
MAQINNMAQGIQTLNPNSNFHKPQVPKSSSFLVFGSKKLKNSANSMLVL
KKDSIFMQLFCSFRISASVATAC; (SEQ ID NO: 31)
MAALVTSQLATSGTVLSVTDRFRRPGFQGLRPRNPADAALGMRTVGASA
APKQSRKPHRFDRRCLSMVV; (SEQ ID NO: 32)
MAALTTSQLATSATGFGIADRSAPSSLLRHGFQGLKPRSPAGGDATSLS
VTTSARATPKQQRSVQRGSRRFPSVVVC; (SEQ ID NO: 33)
MASSVLSSAAVATRSNVAQANMVAPFTGLKSAASFPVSRKQNLDITSIA SNGGRVQC; (SEQ ID
NO: 34) MESLAATSVFAPSRVAVPAARALVRAGTVVPTRRTSSTSGTSGVKCSAA
VTPQASPVISRSAAAA; and (SEQ ID NO: 35)
MGAAATSMQSLKFSNRLVPPSRRLSPVPNNVTCNNLPKSAAPVRTVKCC
ASSWNSTINGAAATTNGASAASS.
[0170] In some case, a Cas12J fusion polypeptide of the present
disclosure comprises: a) a Cas12J polypeptide of the present
disclosure; and b) a chloroplast transit peptide. Thus, for
example, a Cas12J polypeptide/guide RNA complex can be targeted to
the chloroplast. In some cases, this targeting may be achieved by
the presence of an N-terminal extension, called a chloroplast
transit peptide (CTP) or plastid transit peptide. Chromosomal
transgenes from bacterial sources must have a sequence encoding a
CTP sequence fused to a sequence encoding an expressed polypeptide
if the expressed polypeptide is to be compartmentalized in the
plant plastid (e.g. chloroplast). Accordingly, localization of an
exogenous polypeptide to a chloroplast is often 1 accomplished by
means of operably linking a polynucleotide sequence encoding a CTP
sequence to the 5' region of a polynucleotide encoding the
exogenous polypeptide. The CTP is removed in a processing step
during translocation into the plastid. Processing efficiency may,
however, be affected by the amino acid sequence of the CTP and
nearby sequences at the amino terminus (NH.sub.2 terminus) of the
peptide. Other options for targeting to the chloroplast which have
been described are the maize cab-m7 signal sequence (U.S. Pat. No.
7,022,896, WO 97/41228) a pea glutathione reductase signal sequence
(WO 97/41228) and the CTP described in US2009029861.
[0171] In some cases, a Cas12J fusion polypeptide of the present
disclosure can comprise: a) a Cas12J polypeptide of the present
disclosure; and b) an endosomal escape peptide. In some cases, an
endosomal escape polypeptide comprises the amino acid sequence
GLFXALLXLLXSLWXLLLXA (SEQ ID NO: 36), wherein each X is
independently selected from lysine, histidine, and arginine. In
some cases, an endosomal escape polypeptide comprises the amino
acid sequence GLFHALLHLLHSLWHLLLHA (SEQ ID NO: 37).
[0172] For examples of some of the above fusion partners (and more)
used in the context of fusions with Cas9, Zinc Finger, and/or TALE
proteins (for site specific target nucleic modification, modulation
of transcription, and/or target protein modification, e.g., histone
modification), see, e.g.: Nomura et al, J Am Chem Soc. 2007 Jul.
18; 129(28):8676-7; Rivenbark et al., Epigenetics. 2012 April;
7(4):350-60; Nucleic Acids Res. 2016 Jul. 8; 44(12):5615-28;
Gilbert et al., Cell. 2013 Jul. 18; 154(2):442-51; Kearns et al.,
Nat Methods. 2015 May; 12(5):401-3; Mendenhall et al., Nat
Biotechnol. 2013 December; 31(12):1133-6; Hilton et al., Nat
Biotechnol. 2015 May; 33(5):510-7; Gordley et al., Proc Natl Acad
Sci USA. 2009 Mar. 31; 106(13):5053-8; Akopian et al., Proc Natl
Acad Sci USA. 2003 Jul. 22; 100(15):8688-91; Tan et., al., J Virol.
2006 February; 80(4):1939-48; Tan et al., Proc Natl Acad Sci USA.
2003 Oct. 14; 100(21):11997-2002; Papworth et al., Proc Natl Acad
Sci USA. 2003 Feb. 18; 100(4):1621-6; Sanjana et al., Nat Protoc.
2012 Jan. 5; 7(1):171-92; Beerli et al., Proc Natl Acad Sci USA.
1998 Dec. 8; 95(25):14628-33; Snowden et al., Curr Biol. 2002 Dec.
23; 12(24):2159-66; Xu et. al., Xu et al., Cell Discov. 2016 May 3;
2:16009; Komor et al., Nature. 2016 Apr. 20; 533(7603):420-4;
Chaikind et al., Nucleic Acids Res. 2016 Aug. 11; Choudhury at.
al., Oncotarget. 2016 Jun. 23; Du et al., Cold Spring Harb Protoc.
2016 Jan. 4; Pham et al., Methods Mol Biol. 2016; 1358:43-57;
Balboa et al., Stem Cell Reports. 2015 Sep. 8; 5(3):448-59; Hara et
al., Sci Rep. 2015 Jun. 9; 5:11221; Piatek et al., Plant Biotechnol
J. 2015 May; 13(4):578-89; Hu et al., Nucleic Acids Res. 2014
April; 42(7):4375-90; Cheng et al., Cell Res. 2013 October;
23(10):1163-71; and Maeder et al., Nat Methods. 2013 October;
10(10):977-9.
[0173] Additional suitable heterologous polypeptides include, but
are not limited to, a polypeptide that directly and/or indirectly
provides for increased or decreased transcription and/or
translation of a target nucleic acid (e.g., a transcription
activator or a fragment thereof, a protein or fragment thereof that
recruits a transcription activator, a small
molecule/drug-responsive transcription and/or translation
regulator, a translation-regulating protein, etc.). Non-limiting
examples of heterologous polypeptides to accomplish increased or
decreased transcription include transcription activator and
transcription repressor domains. In some such cases, a fusion
Cas12J polypeptide is targeted by the guide nucleic acid (guide
RNA) to a specific location (i.e., sequence) in the target nucleic
acid and exerts locus-specific regulation such as blocking RNA
polymerase binding to a promoter (which selectively inhibits
transcription activator function), and/or modifying the local
chromatin status (e.g., when a fusion sequence is used that
modifies the target nucleic acid or modifies a polypeptide
associated with the target nucleic acid). In some cases, the
changes are transient (e.g., transcription repression or
activation). In some cases, the changes are inheritable (e.g., when
epigenetic modifications are made to the target nucleic acid or to
proteins associated with the target nucleic acid, e.g., nucleosomal
histones).
[0174] Non-limiting examples of heterologous polypeptides for use
when targeting ssRNA target nucleic acids include (but are not
limited to): splicing factors (e.g., RS domains); protein
translation components (e.g., translation initiation, elongation,
and/or release factors; e.g., eIF4G); RNA methylases; RNA editing
enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on
RNA (ADAR), including A to I and/or C to U editing enzymes);
helicases; RNA-binding proteins; and the like. It is understood
that a heterologous polypeptide can include the entire protein or
in some cases can include a fragment of the protein (e.g., a
functional domain).
[0175] The heterologous polypeptide of a subject fusion Cas12J
polypeptide can be any domain capable of interacting with ssRNA
(which, for the purposes of this disclosure, includes
intramolecular and/or intermolecular secondary structures, e.g.,
double-stranded RNA duplexes such as hairpins, stem-loops, etc.),
whether transiently or irreversibly, directly or indirectly,
including but not limited to an effector domain selected from the
group comprising; Endonucleases (for example RNase III, the CRR22
DYW domain, Dicer, and PIN (PilT N-terminus) domains from proteins
such as SMG5 and SMG6); proteins and protein domains responsible
for stimulating RNA cleavage (for example CPSF, CstF, CFIm and
CFIIm); Exonucleases (for example XRN-1 or Exonuclease T);
Deadenylases (for example HNT3); proteins and protein domains
responsible for nonsense mediated RNA decay (for example UPF1,
UPF2, UPF3, UPF3b, RNP S1, Y14, DEK, REF2, and SRm160); proteins
and protein domains responsible for stabilizing RNA (for example
PABP); proteins and protein domains responsible for repressing
translation (for example Ago2 and Ago4); proteins and protein
domains responsible for stimulating translation (for example
Staufen); proteins and protein domains responsible for (e.g.,
capable of) modulating translation (e.g., translation factors such
as initiation factors, elongation factors, release factors, etc.,
e.g., eIF4G); proteins and protein domains responsible for
polyadenylation of RNA (for example PAP1, GLD-2, and Star-PAP);
proteins and protein domains responsible for polyuridinylation of
RNA (for example CI Dl and terminal uridylate transferase);
proteins and protein domains responsible for RNA localization (for
example from IMP1, ZBP1, She2p, She3p, and Bicaudal-D); proteins
and protein domains responsible for nuclear retention of RNA (for
example Rrp6); proteins and protein domains responsible for nuclear
export of RNA (for example TAP, NXF1, THO, TREX, REF, and Aly);
proteins and protein domains responsible for repression of RNA
splicing (for example PTB, Sam68, and hnRNP A1); proteins and
protein domains responsible for stimulation of RNA splicing (for
example Serine/Arginine-rich (SR) domains); proteins and protein
domains responsible for reducing the efficiency of transcription
(for example FUS (TLS)); and proteins and protein domains
responsible for stimulating transcription (for example CDK7 and HIV
Tat). Alternatively, the effector domain may be selected from the
group comprising Endonucleases; proteins and protein domains
capable of stimulating RNA cleavage; Exonucleases; Deadenylases;
proteins and protein domains having nonsense mediated RNA decay
activity; proteins and protein domains capable of stabilizing RNA;
proteins and protein domains capable of repressing translation;
proteins and protein domains capable of stimulating translation;
proteins and protein domains capable of modulating translation
(e.g., translation factors such as initiation factors, elongation
factors, release factors, etc., e.g., eIF4G); proteins and protein
domains capable of polyadenylation of RNA; proteins and protein
domains capable of polyuridinylation of RNA; proteins and protein
domains having RNA localization activity; proteins and protein
domains capable of nuclear retention of RNA; proteins and protein
domains having RNA nuclear export activity; proteins and protein
domains capable of repression of RNA splicing; proteins and protein
domains capable of stimulation of RNA splicing; proteins and
protein domains capable of reducing the efficiency of
transcription; and proteins and protein domains capable of
stimulating transcription. Another suitable heterologous
polypeptide is a PUF RNA-binding domain, which is described in more
detail in WO2012068627, which is hereby incorporated by reference
in its entirety.
[0176] Some RNA splicing factors that can be used (in whole or as
fragments thereof) as heterologous polypeptides for a fusion Cas12J
polypeptide have modular organization, with separate
sequence-specific RNA binding modules and splicing effector
domains. For example, members of the Serine/Arginine-rich (SR)
protein family contain N-terminal RNA recognition motifs (RRMs)
that bind to exonic splicing enhancers (ESEs) in pre-mRNAs and
C-terminal RS domains that promote exon inclusion. As another
example, the hnRNP protein hnRNP Al binds to exonic splicing
silencers (ESSs) through its RRM domains and inhibits exon
inclusion through a C-terminal Glycine-rich domain. Some splicing
factors can regulate alternative use of splice site (ss) by binding
to regulatory sequences between the two alternative sites. For
example, ASF/SF2 can recognize ESEs and promote the use of intron
proximal sites, whereas hnRNP Al can bind to ESSs and shift
splicing towards the use of intron distal sites. One application
for such factors is to generate ESFs that modulate alternative
splicing of endogenous genes, particularly disease associated
genes. For example, Bcl-x pre-mRNA produces two splicing isoforms
with two alternative 5' splice sites to encode proteins of opposite
functions. The long splicing isoform Bcl-xL is a potent apoptosis
inhibitor expressed in long-lived postmitotic cells and is
up-regulated in many cancer cells, protecting cells against
apoptotic signals. The short isoform Bel-xS is a pro-apoptotic
isoform and expressed at high levels in cells with a high turnover
rate (e.g., developing lymphocytes). The ratio of the two Bcl-x
splicing isoforms is regulated by multiple c{acute over
(.omega.)}-elements that are located in either the core exon region
or the exon extension region (i.e., between the two alternative 5'
splice sites). For more examples, see WO2010075303, which is hereby
incorporated by reference in its entirety.
[0177] Further suitable fusion partners include, but are not
limited to, proteins (or fragments thereof) that are boundary
elements (e.g., CTCF), proteins and fragments thereof that provide
periphery recruitment (e.g., Lamin A, Lamin B, etc.), protein
docking elements (e.g., FKBP/FRB, Pil1/Aby1, etc.).
Nucleases
[0178] In some cases, a subject fusion Cas12J polypeptide
comprises: i) a Cas12J polypeptide of the present disclosure; and
ii) a heterologous polypeptide (a "fusion partner"), where the
heterologous polypeptide is a nuclease. Suitable nucleases include,
but are not limited to, a homing nuclease polypeptide; a FokI
polypeptide; a transcription activator-like effector nuclease
(TALEN) polypeptide; a MegaTAL polypeptide; a meganuclease
polypeptide; a zinc finger nuclease (ZFN); an ARCUS nuclease; and
the like. The meganuclease can be engineered from an LADLIDADG
homing endonuclease (LHE). A megaTAL polypeptide can comprise a
TALE DNA binding domain and an engineered meganuclease. See, e.g.,
WO 2004/067736 (homing endonuclease); Urnov et al. (2005) Nature
435:646 (ZFN); Mussolino et al. (2011) Nucle. Acids Res. 39:9283
(TALE nuclease); Boissel et al. (2013) Nucl. Acids Res. 42:2591
(MegaTAL).
Reverse Transcriptases
[0179] In some cases, a subject fusion Cas12J polypeptide
comprises: i) a Cas12J polypeptide of the present disclosure; and
ii) a heterologous polypeptide (a "fusion partner"), where the
heterologous polypeptide is a reverse transcriptase polypeptide. In
some cases, the Cas12J polypeptide is catalytically inactive.
Suitable reverse transcriptases include, e.g., a murine leukemia
virus reverse transcriptase; a Rous sarcoma virus reverse
transcriptase; a human immunodeficiency virus type I reverse
transcriptase; a Moloney murine leukemia virus reverse
transcriptase; and the like.
Base Editors
[0180] In some cases, a Cas12J fusion polypeptide of the present
disclosure comprises: i) a Cas12J polypeptide of the present
disclosure; and ii) a heterologous polypeptide (a "fusion
partner"), where the heterologous polypeptide is a base editor.
Suitable base editors include, e.g., an adenosine deaminase; a
cytidine deaminase (e.g., an activation-induced cytidine deaminase
(AID)); APOBEC3G; and the like); and the like.
[0181] A suitable adenosine deaminase is any enzyme that is capable
of deaminating adenosine in DNA. In some cases, the deaminase is a
TadA deaminase.
[0182] In some cases, a suitable adenosine deaminase comprises an
amino acid sequence having at least 80%, at least 85%, at least
90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following amino acid sequence:
TABLE-US-00002 (SEQ ID NO: 38)
MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPI
GRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSR
IGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSD
FFRMRRQEIKAQKKAQSSTD
[0183] In some cases, a suitable adenosine deaminase comprises an
amino acid sequence having at least 80%, at least 85%, at least
90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following amino acid sequence:
TABLE-US-00003 (SEQ ID NO: 39)
MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNN
RVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPC
VMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGI
LADECAALLSDFFRMRRQEIKAQKKAQSSTD.
[0184] In some cases, a suitable adenosine deaminase comprises an
amino acid sequence having at least 80%, at least 85%, at least
90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following Staphylococcus aureus TadA amino
acid sequence:
TABLE-US-00004 (SEQ ID NO: 40)
MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRE
TLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSR
IPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTT
FFKNLRANKKSTN:
[0185] In some cases, a suitable adenosine deaminase comprises an
amino acid sequence having at least 80%, at least 85%, at least
90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following Bacillus subtilis TadA amino
acid sequence:
TABLE-US-00005 (SEQ ID NO: 41)
MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQR
SIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKV
VFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRE
LRKKKKAARKNLSE
[0186] In some cases, a suitable adenosine deaminase comprises an
amino acid sequence having at least 80%, at least 85%, at least
90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following Salmonella typhimurium TadA:
TABLE-US-00006 (SEQ ID NO: 42)
MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNH
RVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPC
VMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGV
LRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV
[0187] In some cases, a suitable adenosine deaminase comprises an
amino acid sequence having at least 80%, at least 85%, at least
90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following Shewanella putrefaciens TadA
amino acid sequence:
TABLE-US-00007 (SEQ ID NO: 43)
MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPT
AHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVY
GARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRR
DEKKALKLAQRAQQGIE
[0188] In some cases, a suitable adenosine deaminase comprises an
amino acid sequence having at least 80%, at least 85%, at least
90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following Haemophilus influenzae F3031
TadA amino acid sequence:
TABLE-US-00008 (SEQ ID NO: 44)
MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGW
NLSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAI
LHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQ
KLSTFFQKRREEKKIEKALLKSLSDK
[0189] In some cases, a suitable adenosine deaminase comprises an
amino acid sequence having at least 80%, at least 85%, at least
90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following Caulobacter crescentus TadA
amino acid sequence:
TABLE-US-00009 (SEQ ID NO: 45)
MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAG
NGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAI
SHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESAD
LLRGFFRARRKAKI
[0190] In some cases, a suitable adenosine deaminase comprises an
amino acid sequence having at least 80%, at least 85%, at least
90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following Geobacter sulfurreducens TadA
amino acid sequence:
TABLE-US-00010 (SEQ ID NO: 46)
MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGH
NLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAI
ILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGT
MLSDFFRDLRRRKKAKATPALFIDERKVPPEP
[0191] Cytidine deaminases suitable for inclusion in a CRISPR/Cas
effector polypeptide fusion polypeptide include any enzyme that is
capable of deaminating cytidine in DNA.
[0192] In some cases, the cytidine deaminase is a deaminase from
the apolipoprotein B mRNA-editing complex (APOBEC) family of
deaminases. In some cases, the APOBEC family deaminase is selected
from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase,
APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase,
APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and
APOBEC3H deaminase. In some cases, the cytidine deaminase is an
activation induced deaminase (AID).
[0193] In some cases, a suitable cytidine deaminase comprises an
amino acid sequence having at least 80%, at least 85%, at least
90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the following amino acid sequence:
TABLE-US-00011 (SEQ ID NO: 47)
MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYL
RNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFL
RGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYC
WNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTL GL
[0194] In some cases, a suitable cytidine deaminase is an AID and
comprises an amino acid sequence having at least 80%, at least 85%,
at least 90%, at least 95%, at least 98%, at least 99%, or 100%,
amino acid sequence identity to the following amino acid sequence:
MDSLLMNRRK
TABLE-US-00012 (SEQ ID NO: 48) FLYQFKNVRW AKGRRETYLC YVVKRRDSAT
SFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTW FTSWSPCYDC ARHVADFLRG
NPNLSLRIFT ARLYFCEDRK AEPEGLRRLH RAGVQIAIMT FKENHERTFK AWEGLHENSV
RLSRQLRRIL LPLYEVDDLR DAFRTLGL.
[0195] In some cases, a suitable cytidine deaminase is an AID and
comprises an amino acid sequence having at least 80%, at least 85%,
at least 90%, at least 95%, at least 98%, at least 99%, or 100%,
amino acid sequence identity to the following amino acid sequence:
MDSLLMNRRK
TABLE-US-00013 (SEQ ID NO: 47) FLYQFKNVRW AKGRRETYLC YVVKRRDSAT
SFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTW FTSWSPCYDC ARHVADFLRG
NPNLSLRIFT ARLYFCEDRK AEPEGLRRLH RAGVQIAIMT FKDYFYCWNT FVENHERTFK
AWEGLHENSV RLSRQLRRIL LPLYEVDDLR DAFRTLGL.
Transcription Factors
[0196] In some cases, a Cas12J fusion polypeptide of the present
disclosure comprises: i) a Cas12J polypeptide of the present
disclosure; and ii) a heterologous polypeptide (a "fusion
partner"), where the heterologous polypeptide is a transcription
factor. A transcription factor can include: i) a DNA binding
domain; and ii) a transcription activator. A transcription factor
can include: i) a DNA binding domain; and ii) a transcription
repressor. Suitable transcription factors include polypeptides that
include a transcription activator or a transcription repressor
domain (e.g., the Kruppel associated box (KRAB or SKD); the Mad
mSIN3 interaction domain (SID); the ERF repressor domain (ERD),
etc.); zinc-finger-based artificial transcription factors (see,
e.g., Sera (2009) Adv. Drug Deliv. 61:513); TALE-based artificial
transcription factors (see, e.g., Liu et al. (2013) Nat. Rev.
Genetics 14:781); and the like. In some cases, the transcription
factor comprises a VP64 polypeptide (transcriptional activation).
In some cases, the transcription factor comprises a
Kruppel-associated box (KRAB) polypeptide (transcriptional
repression). In some cases, the transcription factor comprises a
Mad mSIN3 interaction domain (SID) polypeptide (transcriptional
repression). In some cases, the transcription factor comprises an
ERF repressor domain (ERD) polypeptide (transcriptional
repression). For example, in some cases, the transcription factor
is a transcriptional activator, where the transcriptional activator
is GAL4-VP16.
Recombinases
[0197] In some cases, a Cas12J fusion polypeptide of the present
disclosure comprises: i) a Cas12J polypeptide of the present
disclosure; and ii) a heterologous polypeptide (a "fusion
partner"), where the heterologous polypeptide is a recombinase.
Suitable recombinases include, e.g., a Cre recombinase; a Hin
recombinase; a Tre recombinase; a FLP recombinase; and the
like.
[0198] Examples of various additional suitable heterologous
polypeptide (or fragments thereof) for a subject fusion Cas12J
polypeptide include, but are not limited to, those described in the
following applications (which publications are related to other
CRISPR endonucleases such as Cas9, but the described fusion
partners can also be used with Cas12J instead): PCT patent
applications: WO2010075303, WO2012068627, and WO2013155555, and can
be found, for example, in U.S. patents and patent applications:
U.S. Pat. Nos. 8,906,616; 8,895,308; 8,889,418; 8,889,356;
8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797;
20140170753; 20140179006; 20140179770; 20140186843; 20140186919;
20140186958; 20140189896; 20140227787; 20140234972; 20140242664;
20140242699; 20140242700; 20140242702; 20140248702; 20140256046;
20140273037; 20140273226; 20140273230; 20140273231; 20140273232;
20140273233; 20140273234; 20140273235; 20140287938; 20140295556;
20140295557; 20140298547; 20140304853; 20140309487; 20140310828;
20140310830; 20140315985; 20140335063; 20140335620; 20140342456;
20140342457; 20140342458; 20140349400; 20140349405; 20140356867;
20140356956; 20140356958; 20140356959; 20140357523; 20140357530;
20140364333; and 20140377868; all of which are hereby incorporated
by reference in their entirety.
[0199] In some cases, a heterologous polypeptide (a fusion partner)
provides for subcellular localization, i.e., the heterologous
polypeptide contains a subcellular localization sequence (e.g., a
nuclear localization signal (NLS) for targeting to the nucleus, a
sequence to keep the fusion protein out of the nucleus, e.g., a
nuclear export sequence (NES), a sequence to keep the fusion
protein retained in the cytoplasm, a mitochondrial localization
signal for targeting to the mitochondria, a chloroplast
localization signal for targeting to a chloroplast, an ER retention
signal, and the like). In some cases, a Cas12J fusion polypeptide
does not include an NLS so that the protein is not targeted to the
nucleus (which can be advantageous, e.g., when the target nucleic
acid is an RNA that is present in the cytosol). In some cases, the
heterologous polypeptide can provide a tag (i.e., the heterologous
polypeptide is a detectable label) for ease of tracking and/or
purification (e.g., a fluorescent protein, e.g., green fluorescent
protein (GFP), yellow fluorescent protein (YFP), red fluorescent
protein (RFP), cyan fluorescent protein (CFP), mCherry, tdTomato,
and the like; a histidine tag, e.g., a 6.times.His tag; a
hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like).
[0200] In some cases, a Cas12J protein (e.g., a wild type Cas12J
protein, a variant Cas12J protein, a fusion Cas12J protein, a
dCas12J protein, and the like) includes (is fused to) a nuclear
localization signal (NLS) (e.g., in some cases 2 or more, 3 or
more, 4 or more, or 5 or more NLSs). Thus, in some cases, a Cas12J
polypeptide includes one or more NLSs (e.g., 2 or more, 3 or more,
4 or more, or 5 or more NLSs). In some cases, one or more NLSs (2
or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at
or near (e.g., within 50 amino acids of) the N-terminus and/or the
C-terminus. In some cases, one or more NLSs (2 or more, 3 or more,
4 or more, or 5 or more NLSs) are positioned at or near (e.g.,
within 50 amino acids of) the N-terminus. In some cases, one or
more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are
positioned at or near (e.g., within 50 amino acids of) the
C-terminus. In some cases, one or more NLSs (3 or more, 4 or more,
or 5 or more NLSs) are positioned at or near (e.g., within 50 amino
acids of) both the N-terminus and the C-terminus. In some cases, an
NLS is positioned at the N-terminus and an NLS is positioned at the
C-terminus.
[0201] In some cases, a Cas12J protein (e.g., a wild type Cas12J
protein, a variant Cas12J protein, a fusion Cas12J protein, a
dCas12J protein, and the like) includes (is fused to) between 1 and
10 NLSs (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6,
or 2-5 NLSs). In some cases, a Cas12J protein (e.g., a wild type
Cas12J protein, a variant Cas12J protein, a fusion Cas12J protein,
a dCas12J protein, and the like) includes (is fused to) between 2
and 5 NLSs (e.g., 2-4, or 2-3 NLSs).
[0202] Non-limiting examples of NLSs include an NLS sequence
derived from: the NLS of the SV40 virus large T-antigen, having the
amino acid sequence PKKKRKV (SEQ ID NO: 49); the NLS from
nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the
sequence KRPAATKKAGQAKKKK (SEQ ID NO: 50)); the c-myc NLS having
the amino acid sequence PAAKRVKLD (SEQ ID NO: 51) or RQRRNELKRSP
(SEQ ID NO: 52); the hRNPA1 M9 NLS having the sequence
NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 53); the
sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 54)
of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ
ID NO: 55) and PPKKARED (SEQ ID NO: 98) of the myoma T protein; the
sequence PQPKKKPL (SEQ ID NO: 56) of human p53; the sequence
SALIKKKKKMAP (SEQ ID NO: 57) of mouse c-abl IV; the sequences DRLRR
(SEQ ID NO: 58) and PKQKKRK (SEQ ID NO: 59) of the influenza virus
NS1; the sequence RKLKKKIKKL (SEQ ID NO: 60) of the Hepatitis virus
delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 61) of the mouse
M.times.1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:
62) of the human poly(ADP-ribose) polymerase; and the sequence
RKCLQAGMNLEARKTKK (SEQ ID NO: 63) of the steroid hormone receptors
(human) glucocorticoid. In general, NLS (or multiple NLSs) are of
sufficient strength to drive accumulation of the Cas12J protein in
a detectable amount in the nucleus of a eukaryotic cell. Detection
of accumulation in the nucleus may be performed by any suitable
technique. For example, a detectable marker may be fused to the
Cas12J protein such that location within a cell may be visualized.
Cell nuclei may also be isolated from cells, the contents of which
may then be analyzed by any suitable process for detecting protein,
such as immunohistochemistry, Western blot, or enzyme activity
assay. Accumulation in the nucleus may also be determined
indirectly.
[0203] In some cases, a Cas12J fusion polypeptide includes a
"Protein Transduction Domain" or PTD (also known as a CPP--cell
penetrating peptide), which refers to a polypeptide,
polynucleotide, carbohydrate, or organic or inorganic compound that
facilitates traversing a lipid bilayer, micelle, cell membrane,
organelle membrane, or vesicle membrane. A PTD attached to another
molecule, which can range from a small polar molecule to a large
macromolecule and/or a nanoparticle, facilitates the molecule
traversing a membrane, for example going from extracellular space
to intracellular space, or cytosol to within an organelle. In some
embodiments, a PTD is covalently linked to the amino terminus a
polypeptide (e.g., linked to a wild type Cas12J to generate a
fusion protein, or linked to a variant Cas12J protein such as a
dCas12J, nickase Cas12J, or fusion Cas12J protein, to generate a
fusion protein). In some embodiments, a PTD is covalently linked to
the carboxyl terminus of a polypeptide (e.g., linked to a wild type
Cas12J to generate a fusion protein, or linked to a variant Cas12J
protein such as a dCas12J, nickase Cas12J, or fusion Cas12J protein
to generate a fusion protein). In some cases, the PTD is inserted
internally in the Cas12J fusion polypeptide (i.e., is not at the N-
or C-terminus of the Cas12J fusion polypeptide) at a suitable
insertion site. In some cases, a subject Cas12J fusion polypeptide
includes (is conjugated to, is fused to) one or more PTDs (e.g.,
two or more, three or more, four or more PTDs). In some cases, a
PTD includes a nuclear localization signal (NLS) (e.g., in some
cases 2 or more, 3 or more, 4 or more, or 5 or more NLSs). Thus, in
some cases, a Cas12J fusion polypeptide includes one or more NLSs
(e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs). In some
embodiments, a PTD is covalently linked to a nucleic acid (e.g., a
Cas12J guide nucleic acid, a polynucleotide encoding a Cas12J guide
nucleic acid, a polynucleotide encoding a Cas12J fusion
polypeptide, a donor polynucleotide, etc.). Examples of PTDs
include but are not limited to a minimal undecapeptide protein
transduction domain (corresponding to residues 47-57 of HIV-1 TAT
comprising YGRKKRRQRRR; SEQ ID NO: 64); a polyarginine sequence
comprising a number of arginines sufficient to direct entry into a
cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22
domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an
Drosophila Antennapedia protein transduction domain (Noguchi et al.
(2003) Diabetes 52(7):1732-1737); a truncated human calcitonin
peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256);
polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA
97:13003-13008); RRQRRTSKLMKR (SEQ ID NO: 65); Transportan
GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 66);
KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 67); and
RQIKIWFQNRRMKWKK (SEQ ID NO: 68). Exemplary PTDs include but are
not limited to, YGRKKRRQRRR (SEQ ID NO: 64), RKKRRQRRR (SEQ ID NO:
70); an arginine homopolymer of from 3 arginine residues to 50
arginine residues; Exemplary PTD domain amino acid sequences
include, but are not limited to, any of the following: YGRKKRRQRRR
(SEQ ID NO: 64); RKKRRQRR (SEQ ID NO: 70); YARAAARQARA (SEQ ID NO:
71); THRLPRRRRRR (SEQ ID NO: 72); and GGRRARRRRRR (SEQ ID NO: 73).
In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera
et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPPs
comprise a polycationic CPP (e.g., Arg9 or "R9") connected via a
cleavable linker to a matching polyanion (e.g., Glu9 or "E9"),
which reduces the net charge to nearly zero and thereby inhibits
adhesion and uptake into cells. Upon cleavage of the linker, the
polyanion is released, locally unmasking the polyarginine and its
inherent adhesiveness, thus "activating" the ACPP to traverse the
membrane.
Linkers (e.g., for Fusion Partners)
[0204] In some embodiments, a subject Cas12J protein can fused to a
fusion partner via a linker polypeptide (e.g., one or more linker
polypeptides). The linker polypeptide may have any of a variety of
amino acid sequences. Proteins can be joined by a spacer peptide,
generally of a flexible nature, although other chemical linkages
are not excluded. Suitable linkers include polypeptides of between
4 amino acids and 40 amino acids in length, or between 4 amino
acids and 25 amino acids in length. These linkers can be produced
by using synthetic, linker-encoding oligonucleotides to couple the
proteins, or can be encoded by a nucleic acid sequence encoding the
fusion protein. Peptide linkers with a degree of flexibility can be
used. The linking peptides may have virtually any amino acid
sequence, bearing in mind that the preferred linkers will have a
sequence that results in a generally flexible peptide. The use of
small amino acids, such as glycine and alanine, are of use in
creating a flexible peptide. The creation of such sequences is
routine to those of skill in the art. A variety of different
linkers are commercially available and are considered suitable for
use.
[0205] Examples of linker polypeptides include glycine polymers
(G).sub.n, glycine-serine polymers (including, for example,
(GS).sub.n, GSGGS.sub.n (SEQ ID NO: 74), GGSGGS.sub.n (SEQ ID NO:
75), and GGGS.sub.n (SEQ ID NO: 76), where n is an integer of at
least one), glycine-alanine polymers, alanine-serine polymers.
Exemplary linkers can comprise amino acid sequences including, but
not limited to, GGSG (SEQ ID NO: 77), GGSGG (SEQ ID NO: 78), GSGSG
(SEQ ID NO: 79), GSGGG (SEQ ID NO: 80), GGGSG (SEQ ID NO: 81),
GSSSG (SEQ ID NO: 82), and the like. The ordinarily skilled artisan
will recognize that design of a peptide conjugated to any desired
element can include linkers that are all or partially flexible,
such that the linker can include a flexible linker as well as one
or more portions that confer less flexible structure.
Detectable Labels
[0206] In some cases, a Cas12J polypeptide of the present
disclosure comprises a detectable label. Suitable detectable labels
and/or moieties that can provide a detectable signal can include,
but are not limited to, an enzyme, a radioisotope, a member of a
specific binding pair; a fluorophore; a fluorescent protein; a
quantum dot; and the like.
[0207] Suitable fluorescent proteins include, but are not limited
to, green fluorescent protein (GFP) or variants thereof, blue
fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP
(CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP
(EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald,
Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP
(dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP),
mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed,
DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFP1,
pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and
kindling protein, Phycobiliproteins and Phycobiliprotein conjugates
including B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin.
Other examples of fluorescent proteins include mHoneydew, mBanana,
mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry,
mGrape1, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat.
Methods 2:905-909), and the like. Any of a variety of fluorescent
and colored proteins from Anthozoan species, as described in, e.g.,
Matz et al. (1999) Nature Biotechnol. 17:969-973, is suitable for
use.
[0208] Suitable enzymes include, but are not limited to, horse
radish peroxidase (HRP), alkaline phosphatase (AP),
beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase,
beta-N-acetylglucosaminidase, .beta.-glucuronidase, invertase,
Xanthine Oxidase, firefly luciferase, glucose oxidase (GO), and the
like.
Protospacer Adjacent Motif (PAM)
[0209] A Cas12J protein binds to target DNA at a target sequence
defined by the region of complementarity between the DNA-targeting
RNA and the target DNA. As is the case for many CRISPR
endonucleases, site-specific binding (and/or cleavage) of a double
stranded target DNA occurs at locations determined by both (i)
base-pairing complementarity between the guide RNA and the target
DNA; and (ii) a short motif [referred to as the protospacer
adjacent motif (PAM)] in the target DNA.
[0210] In some embodiments, the PAM for a Cas12J protein is
immediately 5' of the target sequence of the non-complementary
strand of the target DNA (the complementary strand: (i) hybridizes
to the guide sequence of the guide RNA, while the non-complementary
strand does not directly hybridize with the guide RNA; and (ii) is
the reverse complement of the non-complementary strand).
[0211] In some cases (e.g., when Cas12J-1947455--also referred to
herein as "ortholog #1"--as described herein is used), the PAM
sequence of the non-complementary strand is 5'-VTTR-3' (where V is
G, A, or C and R is A or G)--see, e.g., FIG. 13A. Thus, in some
cases, suitable PAMs can include GTTA, GTTG, ATTA, ATTG, CTTA, and
CTTG.
[0212] In some cases (e.g., when Cas12J-2071242--also referred to
herein as "ortholog #2"--as described herein is used), the PAM
sequence of the non-complementary strand is 5'-TBN-3' (where B is
T, C, or G)--see, e.g., FIG. 13A. Thus, in some cases, suitable
PAMs can include TTA, TTC, TTT, TTG, TCA, TCC, TCT, TCG, TGA, TGC,
TGT, and TGG. In some embodiments (e.g., when Cas12J-2071242--also
referred to herein as "ortholog #2"--as described herein is used),
the PAM sequence of the non-complementary strand is 5'-TNN-3'.
[0213] In some cases (e.g., when Cas12J-3339380--also referred to
herein as "ortholog #3"--as described herein is used), the PAM
sequence of the non-complementary strand is 5'-VTTB-3' (where V is
G, A, or C and where B is T, C, or G)--see, e.g., FIG. 13A. Thus,
in some cases, suitable PAMs can include GTTT, GTTC, GTTG, ATTT,
ATTC, ATTG, CTTT, CTTC, CTTG. In some cases (e.g., when
Cas12J-3339380--also referred to herein as "ortholog #3"--as
described herein is used), the PAM sequence of the
non-complementary strand is 5'-NTTN-3'. In some cases (e.g., when
Cas12J-3339380--also referred to herein as "ortholog #3"--as
described herein is used), the PAM sequence of the
non-complementary strand is 5'-VTTN-3' (where V is G, A, or C). In
some embodiments (e.g., when Cas12J-3339380--also referred to
herein as "ortholog #3"--as described herein is used), the PAM
sequence of the non-complementary strand is 5'-VTTC-3'.
[0214] In some cases, different Cas12J proteins (i.e., Cas12J
proteins from various species) may be advantageous to use in the
various provided methods in order to capitalize on various
enzymatic characteristics of the different Cas12J proteins (e.g.,
for different PAM sequence preferences; for increased or decreased
enzymatic activity; for an increased or decreased level of cellular
toxicity; to change the balance between NHEJ, homology-directed
repair, single strand breaks, double strand breaks, etc.; to take
advantage of a short total sequence; and the like). Cas12J proteins
from different species may require different PAM sequences in the
target DNA. Thus, for a particular Cas12J protein of choice, the
PAM sequence preference may be different than the sequences
described above. Various methods (including in silico and/or wet
lab methods) for identification of the appropriate PAM sequence are
known in the art and are routine, and any convenient method can be
used. For example, PAM sequences described herein were identified
using a PAM depletion assay (e.g., see working examples below), but
could also have been identified using a variety of different
methods (including computational analysis of sequencing data--as
known in the art).
Cas12J Guide RNA
[0215] A nucleic acid that binds to a Cas12J protein, forming a
ribonucleoprotein complex (RNP), and targets the complex to a
specific location within a target nucleic acid (e.g., a target DNA)
is referred to herein as a "Cas12J guide RNA" or simply as a "guide
RNA." It is to be understood that in some cases, a hybrid DNA/RNA
can be made such that a Cas12J guide RNA includes DNA bases in
addition to RNA bases, but the term "Cas12J guide RNA" is still
used to encompass such a molecule herein.
[0216] A Cas12J guide RNA can be said to include two segments, a
targeting segment and a protein-binding segment. The
protein-binding segment is also referred to herein as the "constant
region" of the guide RNA. The targeting segment of a Cas12J guide
RNA includes a nucleotide sequence (a guide sequence) that is
complementary to (and therefore hybridizes with) a specific
sequence (a target site) within a target nucleic acid (e.g., a
target dsDNA, a target ssRNA, a target ssDNA, the complementary
strand of a double stranded target DNA, etc.). The protein-binding
segment (or "protein-binding sequence") interacts with (binds to) a
Cas12J polypeptide.
[0217] The protein-binding segment of a subject Cas12J guide RNA
can include two complementary stretches of nucleotides that
hybridize to one another to form a double stranded RNA duplex
(dsRNA duplex). Site-specific binding and/or cleavage of a target
nucleic acid (e.g., genomic DNA, ds DNA, RNA, etc.) can occur at
locations (e.g., target sequence of a target locus) determined by
base-pairing complementarity between the Cas12J guide RNA (the
guide sequence of the Cas12J guide RNA) and the target nucleic
acid.
[0218] A Cas12J guide RNA and a Cas12J protein (e.g., a wild-type
Cas12J protein; a variant Cas12J protein; a fusion Cas12J
polypeptide; etc.) form a complex (e.g., bind via non-covalent
interactions). The Cas12J guide RNA provides target specificity to
the complex by including a targeting segment, which includes a
guide sequence (a nucleotide sequence that is complementary to a
sequence of a target nucleic acid). The Cas12J protein of the
complex provides the site-specific activity (e.g., cleavage
activity provided by the Cas12J protein and/or an activity provided
by the fusion partner in the case of a fusion Cas12J protein). In
other words, the Cas12J protein is guided to a target nucleic acid
sequence (e.g. a target sequence) by virtue of its association with
the Cas12J guide RNA.
[0219] The "guide sequence" also referred to as the "targeting
sequence" of a Cas12J guide RNA can be modified so that the Cas12J
guide RNA can target a Cas12J protein (e.g., a naturally occurring
Cas12J protein, a fusion Cas12J polypeptide, and the like) to any
desired sequence of any desired target nucleic acid, with the
exception (e.g., as described herein) that the PAM sequence can be
taken into account. Thus, for example, a Cas12J guide RNA can have
a guide sequence with complementarity to (e.g., can hybridize to) a
sequence in a nucleic acid in a eukaryotic cell, e.g., a viral
nucleic acid, a eukaryotic nucleic acid (e.g., a eukaryotic
chromosome, chromosomal sequence, a eukaryotic RNA, etc.), and the
like.
Guide Sequence of a Cas12J Guide RNA
[0220] A subject Cas12J guide RNA includes a guide sequence (i.e.,
a targeting sequence), which is a nucleotide sequence that is
complementary to a sequence (a target site) in a target nucleic
acid. In other words, the guide sequence of a Cas12J guide RNA can
interact with a target nucleic acid (e.g., double stranded DNA
(dsDNA), single stranded DNA (ssDNA), single stranded RNA (ssRNA),
or double stranded RNA (dsRNA)) in a sequence-specific manner via
hybridization (i.e., base pairing). The guide sequence of a Cas12J
guide RNA can be modified (e.g., by genetic engineering)/designed
to hybridize to any desired target sequence (e.g., while taking the
PAM into account, e.g., when targeting a dsDNA target) within a
target nucleic acid (e.g., a eukaryotic target nucleic acid such as
genomic DNA).
[0221] In some cases, the percent complementarity between the guide
sequence and the target site of the target nucleic acid is 60% or
more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85%
or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or
more, or 100%). In some cases, the percent complementarity between
the guide sequence and the target site of the target nucleic acid
is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or
more, 98% or more, 99% or more, or 100%). In some cases, the
percent complementarity between the guide sequence and the target
site of the target nucleic acid is 90% or more (e.g., 95% or more,
97% or more, 98% or more, 99% or more, or 100%). In some cases, the
percent complementarity between the guide sequence and the target
site of the target nucleic acid is 100%.
[0222] In some cases, the percent complementarity between the guide
sequence and the target site of the target nucleic acid is 100%
over the seven contiguous 3'-most nucleotides of the target site of
the target nucleic acid.
[0223] In some cases, the percent complementarity between the guide
sequence and the target site of the target nucleic acid is 60% or
more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90%
or more, 95% or more, 97% or more, 98% or more, 99% or more, or
100%) over 17 or more (e.g., 18 or more, 19 or more, 20 or more, 21
or more, 22 or more) contiguous nucleotides. In some cases, the
percent complementarity between the guide sequence and the target
site of the target nucleic acid is 80% or more (e.g., 85% or more,
90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or
100%) over 17 or more (e.g., 18 or more, 19 or more, 20 or more, 21
or more, 22 or more) contiguous nucleotides. In some cases, the
percent complementarity between the guide sequence and the target
site of the target nucleic acid is 90% or more (e.g., 95% or more,
97% or more, 98% or more, 99% or more, or 100%) over 17 or more
(e.g., 18 or more, 19 or more, 20 or more, 21 or more, 22 or more)
contiguous nucleotides. In some cases, the percent complementarity
between the guide sequence and the target site of the target
nucleic acid is 100% over 17 or more (e.g., 18 or more, 19 or more,
20 or more, 21 or more, 22 or more) contiguous nucleotides.
[0224] In some cases, the percent complementarity between the guide
sequence and the target site of the target nucleic acid is 60% or
more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90%
or more, 95% or more, 97% or more, 98% or more, 99% or more, or
100%) over 19 or more (e.g., 20 or more, 21 or more, 22 or more)
contiguous nucleotides. In some cases, the percent complementarity
between the guide sequence and the target site of the target
nucleic acid is 80% or more (e.g., 85% or more, 90% or more, 95% or
more, 97% or more, 98% or more, 99% or more, or 100%) over 19 or
more (e.g., 20 or more, 21 or more, 22 or more) contiguous
nucleotides. In some cases, the percent complementarity between the
guide sequence and the target site of the target nucleic acid is
90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or
more, or 100%) over 19 or more (e.g., 20 or more, 21 or more, 22 or
more) contiguous nucleotides. In some cases, the percent
complementarity between the guide sequence and the target site of
the target nucleic acid is 100% over 19 or more (e.g., 20 or more,
21 or more, 22 or more) contiguous nucleotides.
[0225] In some cases, the percent complementarity between the guide
sequence and the target site of the target nucleic acid is 60% or
more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90%
or more, 95% or more, 97% or more, 98% or more, 99% or more, or
100%) over 17-25 contiguous nucleotides. In some cases, the percent
complementarity between the guide sequence and the target site of
the target nucleic acid is 80% or more (e.g., 85% or more, 90% or
more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%)
over 17-25 contiguous nucleotides. In some cases, the percent
complementarity between the guide sequence and the target site of
the target nucleic acid is 90% or more (e.g., 95% or more, 97% or
more, 98% or more, 99% or more, or 100%) over 17-25 contiguous
nucleotides. In some cases, the percent complementarity between the
guide sequence and the target site of the target nucleic acid is
100% over 17-25 contiguous nucleotides.
[0226] In some cases, the percent complementarity between the guide
sequence and the target site of the target nucleic acid is 60% or
more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90%
or more, 95% or more, 97% or more, 98% or more, 99% or more, or
100%) over 19-25 contiguous nucleotides. In some cases, the percent
complementarity between the guide sequence and the target site of
the target nucleic acid is 80% or more (e.g., 85% or more, 90% or
more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%)
over 19-25 contiguous nucleotides. In some cases, the percent
complementarity between the guide sequence and the target site of
the target nucleic acid is 90% or more (e.g., 95% or more, 97% or
more, 98% or more, 99% or more, or 100%) over 19-25 contiguous
nucleotides. In some cases, the percent complementarity between the
guide sequence and the target site of the target nucleic acid is
100% over 19-25 contiguous nucleotides.
[0227] In some cases, the guide sequence has a length in a range of
from 17-30 nucleotides (nt) (e.g., from 17-25, 17-22, 17-20, 19-30,
19-25, 19-22, 19-20, 20-30, 20-25, or 20-22 nt). In some cases, the
guide sequence has a length in a range of from 17-25 nucleotides
(nt) (e.g., from 17-22, 17-20, 19-25, 19-22, 19-20, 20-25, or 20-22
nt). In some cases, the guide sequence has a length of 17 or more
nt (e.g., 18 or more, 19 or more, 20 or more, 21 or more, or 22 or
more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In
some cases, the guide sequence has a length of 19 or more nt (e.g.,
20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22
nt, 23 nt, 24 nt, 25 nt, etc.). In some cases, the guide sequence
has a length of 17 nt. In some cases, the guide sequence has a
length of 18 nt. In some cases, the guide sequence has a length of
19 nt. In some cases, the guide sequence has a length of 20 nt. In
some cases, the guide sequence has a length of 21 nt. In some
cases, the guide sequence has a length of 22 nt. In some cases, the
guide sequence has a length of 23 nt.
[0228] In some cases, the guide sequence (also referred to as a
"spacer sequence") has a length of from 15 to 50 nucleotides (e.g.,
from 15 nucleotides (nt) to 20 nt, from 20 nt to 25 nt, from 25 nt
to 30 nt, from 30 nt to 35 nt, from 35 nt to 40 nt, from 40 nt to
45 nt, or from 45 nt to 50 nt).
Protein-Binding Segment of a Cas12J Guide RNA
[0229] The protein-binding segment (the "constant region") of a
subject Cas12J guide RNA interacts with a Cas12J protein. The
Cas12J guide RNA guides the bound Cas12J protein to a specific
nucleotide sequence within target nucleic acid via the
above-mentioned guide sequence. The protein-binding segment of a
Cas12J guide RNA can include two stretches of nucleotides that are
complementary to one another and hybridize to form a double
stranded RNA duplex (dsRNA duplex). Thus, in some cases, the
protein-binding segment includes a dsRNA duplex. In some cases, the
dsRNA duplex region includes a range of from 5-25 base pairs (bp)
(e.g., from 5-22, 5-20, 5-18, 5-15, 5-12, 5-10, 5-8, 8-25, 8-22,
8-18, 8-15, 8-12, 12-25, 12-22, 12-18, 12-15, 13-25, 13-22, 13-18,
13-15, 14-25, 14-22, 14-18, 14-15, 15-25, 15-22, 15-18, 17-25,
17-22, or 17-18 bp, e.g., 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp,
etc.). In some cases, the dsRNA duplex region includes a range of
from 6-15 base pairs (bp) (e.g., from 6-12, 6-10, or 6-8 bp, e.g.,
6 bp, 7 bp, 8 bp, 9 bp, 10 bp, etc.). In some cases, the duplex
region includes 5 or more bp (e.g., 6 or more, 7 or more, or 8 or
more bp). In some cases, the duplex region includes 6 or more bp
(e.g., 7 or more, or 8 or more bp). In some cases, not all
nucleotides of the duplex region are paired, and therefore the
duplex forming region can include a bulge. The term "bulge" herein
is used to mean a stretch of nucleotides (which can be one
nucleotide) that do not contribute to a double stranded duplex, but
which are surround 5' and 3' by nucleotides that do contribute, and
as such a bulge is considered part of the duplex region. In some
cases, the dsRNA includes 1 or more bulges (e.g., 2 or more, 3 or
more, 4 or more bulges). In some cases, the dsRNA duplex includes 2
or more bulges (e.g., 3 or more, 4 or more bulges). In some cases,
the dsRNA duplex includes 1-5 bulges (e.g., 1-4, 1-3, 2-5, 2-4, or
2-3 bulges).
[0230] Thus, in some cases, the stretches of nucleotides that
hybridize to one another to form the dsRNA duplex have 70%-100%
complementarity (e.g., 75%-100%, 80%-10%, 85%-100%, 90%-100%,
95%-100% complementarity) with one another. In some cases, the
stretches of nucleotides that hybridize to one another to form the
dsRNA duplex have 70%-100% complementarity (e.g., 75%-100%,
80%-10%, 85%-100%, 90%-100%, 95%-100% complementarity) with one
another. In some cases, the stretches of nucleotides that hybridize
to one another to form the dsRNA duplex have 85%-100%
complementarity (e.g., 90%-100%, 95%-100% complementarity) with one
another. In some cases, the stretches of nucleotides that hybridize
to one another to form the dsRNA duplex have 70%-95%
complementarity (e.g., 75%-95%, 80%-95%, 85%-95%, 90%-95%
complementarity) with one another.
[0231] In other words, in some embodiments, the dsRNA duplex
includes two stretches of nucleotides that have 70%-100%
complementarity (e.g., 75%-100%, 80%-10%, 85%-100%, 90%-100%,
95%-100% complementarity) with one another. In some cases, the
dsRNA duplex includes two stretches of nucleotides that have
85%-100% complementarity (e.g., 90%-100%, 95%-100% complementarity)
with one another. In some cases, the dsRNA duplex includes two
stretches of nucleotides that have 70%-95% complementarity (e.g.,
75%-95%, 80%-95%, 85%-95%, 90%-95% complementarity) with one
another.
[0232] The duplex region of a subject Cas12J guide RNA can include
one or more (1, 2, 3, 4, 5, etc) mutations relative to a naturally
occurring duplex region. For example, in some cases a base pair can
be maintained while the nucleotides contributing to the base pair
from each segment can be different. In some cases, the duplex
region of a subject Cas12J guide RNA includes more paired bases,
less paired bases, a smaller bulge, a larger bulge, fewer bulges,
more bulges, or any convenient combination thereof, as compared to
a naturally occurring duplex region (of a naturally occurring
Cas12J guide RNA).
[0233] Examples of various Cas9 guide RNAs can be found in the art,
and in some cases variations similar to those introduced into Cas9
guide RNAs can also be introduced into Cas12J guide RNAs of the
present disclosure (e.g., mutations to the dsRNA duplex region,
extension of the 5' or 3' end for added stability for to provide
for interaction with another protein, and the like). For example,
see Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21;
Chylinski et al., RNA Biol. 2013 May; 10(5):726-37; Ma et al.,
Biomed Res Int. 2013; 2013:270805; Hou et al., Proc Natl Acad Sci
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8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006;
20140179770; 20140186843; 20140186919; 20140186958; 20140189896;
20140227787; 20140234972; 20140242664; 20140242699; 20140242700;
20140242702; 20140248702; 20140256046; 20140273037; 20140273226;
20140273230; 20140273231; 20140273232; 20140273233; 20140273234;
20140273235; 20140287938; 20140295556; 20140295557; 20140298547;
20140304853; 20140309487; 20140310828; 20140310830; 20140315985;
20140335063; 20140335620; 20140342456; 20140342457; 20140342458;
20140349400; 20140349405; 20140356867; 20140356956; 20140356958;
20140356959; 20140357523; 20140357530; 20140364333; and
20140377868; all of which are hereby incorporated by reference in
their entirety.
[0234] Examples of constant regions suitable for inclusion in a
Cas12J guide RNA are provided in FIG. 7 (e.g., where T is
substituted with U). A Cas12J guide RNA can include a constant
region having from 1 to 5 nucleotide substitutions compared to any
one of the nucleotide sequences depicted in FIG. 7. As one example,
the constant region of a Cas12J guide RNA can comprise the
nucleotide sequence: GUCUCGACUAAUCGAGCAAUCGUUUGAGAUCUCUCC (SEQ ID
NO: 83). As another example, the constant region of a Cas12J guide
RNA can comprise the nucleotide sequence:
GUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGAC (SEQ ID NO: 84). As another
example, the constant region of a Cas12J guide RNA can comprise the
nucleotide sequence: GUCCCAGCGUACUGGGCAAUCAAUAGTCGUUUUGGU (SEQ ID
NO: 85). As another example, the constant region of a Cas12J guide
RNA can comprise the nucleotide sequence:
CACAGGAGAGAUCUCAAACGAUUGCUCGAUUAGUCGAGAC (SEQ ID NO: 86). As
another example, the constant region of a Cas12J guide RNA can
comprise the nucleotide sequence:
UAAUGUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGAC (SEQ ID NO: 87). As
another example, the constant region of a Cas12J guide RNA can
comprise the nucleotide sequence:
AUUAACCAAAACGACUAUUGAUUGCCCAGUACGCUGGGAC (SEQ ID NO: 88).
[0235] A Cas12J guide RNA constant region can include any one of
the nucleotide sequences depicted in FIG. 8. A Cas12J guide RNA
constant region can include a nucleotide sequence within the
consensus sequence(s) depicted in FIG. 8.
[0236] The nucleotide sequences (with T substituted with U) can be
combined with a spacer sequence (where the spacer sequence
comprises a target nucleic acid-binding sequence ("guide
sequence")) of choice that is from 15 to 50 nucleotides (e.g., from
15 nucleotides (nt) to 20 nt, from 20 nt to 25 nt, from 25 nt to 30
nt, from 30 nt to 35 nt, from 35 nt to 40 nt, from 40 nt to 45 nt,
or from 45 nt to 50 nt in length). In some cases, the spacer
sequence is 35-38 nucleotides in length. For example, any one of
the nucleotide sequences (with T substituted with U) depicted in
FIG. 7 can be included in a guide RNA comprising (N)n-constant
region, where N is any nucleotide and n is an integer from 15 to 50
(e.g., from 15 to 20, from 20 to 25, from 25 to 30, from 30 to 35,
from 35 to 38, from 35 to 40, from 40 to 45, or from 45 to 50). The
reverse complement of any one of the nucleotide sequences depicted
in FIG. 7 (but with T substituted with U) can be included in a
guide RNA comprising constant region-(N)n, where N is any
nucleotide and n is an integer from 15 to 50 (e.g., from 15 to 20,
from 20 to 25, from 25 to 30, from 30 to 35, from 35 to 38, from 35
to 40, from 40 to 45, or from 45 to 50).
[0237] As one example, a guide RNA can have the following
nucleotide sequence:
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGUCUCGACUAAUCGAGCAA
UCGUUUGAGAUCUCUCC (SEQ ID NO: 89) or in some cases the reverse
complement, where N is any nucleotide, e.g., where the stretch of
Ns includes a target nucleic acid-binding sequence.
[0238] As another example, a guide RNA can have the following
nucleotide sequence:
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGUCGGAACGCUCAACGAUU
GCCCCUCACGAGGGGAC (SEQ ID NO: 90) or in some cases the reverse
complement, where N is any nucleotide, e.g., where the stretch of
Ns includes a target nucleic acid-binding sequence.
[0239] As one example, a guide RNA can have the following
nucleotide sequence: GUCUCGACUAAUCGAGCAAUCGUUUGAGAUCUCUCC-`guide
sequence` (e.g.,
GUCUCGACUAAUCGAGCAAUCGUUUGAGAUCUCUCCNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNN (SEQ ID NO: 91), where the stretch of Ns
represents the guide sequence/targeting sequence and N is any
nucleotide). As another example, a guide RNA can have the following
nucleotide sequence: GGAGAGAUCUCAAACGAUUGCUCGAUUAGUCGAGAC-`guide
sequence` (e.g.,
GGAGAGAUCUCAAACGAUUGCUCGAUUAGUCGAGACNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNN (SEQ ID NO: 92), where the stretch of Ns
represents the guide sequence/targeting sequence and N is any
nucleotide).
[0240] As another example, a guide RNA can have the following
nucleotide sequence: GUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGAC-`guide
sequence` (e.g.,
GUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGACNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNN (SEQ ID NO: 93), where the stretch of Ns
represents the guide sequence/targeting sequence and N is any
nucleotide). As another example, a guide RNA can have the following
nucleotide sequence: GUCCCCUCGUGAGGGGCAAUCGUUGAGCGUUCCGAC-`guide
sequence` (e.g.,
GUCCCCUCGUGAGGGGCAAUCGUUGAGCGUUCCGACNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNN (SEQ ID NO: 94), where the stretch of Ns
represents the guide sequence/targeting sequence and N is any
nucleotide).
[0241] As another example, a guide RNA can have the following
nucleotide sequence:
CACAGGAGAGAUCUCAAACGAUUGCUCGAUUAGUCGAGAC-`guide sequence` (e.g.,
CACAGGAGAGAUCUCAAACGAUUGCUCGAUUAGUCGAGACNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 95), where the stretch of Ns
represents the guide sequence/targeting sequence and N is any
nucleotide). As another example, a guide RNA can have the following
nucleotide sequence:
UAAUGUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGAC-`guide sequence` (e.g.,
UAAUGUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGACNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 96), where the stretch of Ns
represents the guide sequence/targeting sequence and N is any
nucleotide). As another example, a guide RNA can have the following
nucleotide sequence:
AUUAACCAAAACGACUAUUGAUUGCCCAGUACGCUGGGAC-`guide sequence` (e.g.,
AUUAACCAAAACGACUAUUGAUUGCCCAGUACGCUGGGACNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 97), where the stretch of Ns
represents the guide sequence/targeting sequence and N is any
nucleotide).
Cas12J Guide Polynucleotides
[0242] In some cases, a nucleic acid that binds to a Cas12J
protein, forming a nucleic acid/Cas12J polypeptide complex, and
that targets the complex to a specific location within a target
nucleic acid (e.g., a target DNA) comprises ribonucleotides only,
deoxyribonucleotides only, or a mixture of ribonucleotides and
deoxyribonucleotides. In some cases, a guide polynucleotide
comprises ribonucleotides only, and is referred to herein as a
"guide RNA." In some cases, a guide polynucleotide comprises
deoxyribonucleotides only, and is referred to herein as a "guide
DNA." In some cases, a guide polynucleotide comprises both
ribonucleotides and deoxyribonucleotides. A guide polynucleotide
can comprise combinations of ribonucleotide bases,
deoxyribonucleotide bases, nucleotide analogs, modified
nucleotides, and the like; and may further include
naturally-occurring backbone residues and/or linkages and/or
non-naturally-occurring backbone residues and/or linkages.
CAS12J Systems
[0243] The present disclosure provides a Cas12J system. A Cas12J
system of the present disclosure can comprise: a) a Cas12J
polypeptide of the present disclosure and a Cas12J guide RNA; b) a
Cas12J polypeptide of the present disclosure, a Cas12J guide RNA,
and a donor template nucleic acid; c) a Cas12J fusion polypeptide
of the present disclosure and a Cas12J guide RNA; d) a Cas12J
fusion polypeptide of the present disclosure, a Cas12J guide RNA,
and a donor template nucleic acid; e) an mRNA encoding a Cas12J
polypeptide of the present disclosure; and a Cas12J guide RNA; f)
an mRNA encoding a Cas12J polypeptide of the present disclosure, a
Cas12J guide RNA, and a donor template nucleic acid; g) an mRNA
encoding a Cas12J fusion polypeptide of the present disclosure; and
a Cas12J guide RNA; h) an mRNA encoding a Cas12J fusion polypeptide
of the present disclosure, a Cas12J guide RNA, and a donor template
nucleic acid; i) a recombinant expression vector comprising a
nucleotide sequence encoding a Cas12J polypeptide of the present
disclosure and a nucleotide sequence encoding a Cas12J guide RNA;
j) a recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J polypeptide of the present disclosure, a
nucleotide sequence encoding a Cas12J guide RNA, and a nucleotide
sequence encoding a donor template nucleic acid; k) a recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J fusion polypeptide of the present disclosure and a
nucleotide sequence encoding a Cas12J guide RNA; 1) a recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J fusion polypeptide of the present disclosure, a nucleotide
sequence encoding a Cas12J guide RNA, and a nucleotide sequence
encoding a donor template nucleic acid; m) a first recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J polypeptide of the present disclosure, and a second
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J guide RNA; n) a first recombinant expression
vector comprising a nucleotide sequence encoding a Cas12J
polypeptide of the present disclosure, and a second recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J guide RNA; and a donor template nucleic acid; o) a first
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J fusion polypeptide of the present disclosure, and
a second recombinant expression vector comprising a nucleotide
sequence encoding a Cas12J guide RNA; p) a first recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J fusion polypeptide of the present disclosure, and a second
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J guide RNA; and a donor template nucleic acid; q)
a recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J polypeptide of the present disclosure, a
nucleotide sequence encoding a first Cas12J guide RNA, and a
nucleotide sequence encoding a second Cas12J guide RNA; or r) a
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J fusion polypeptide of the present disclosure, a
nucleotide sequence encoding a first Cas12J guide RNA, and a
nucleotide sequence encoding a second Cas12J guide RNA; or some
variation of one of (a) through (r).
Nucleic Acids
[0244] The present disclosure provides one or more nucleic acids
comprising one or more of: a donor polynucleotide sequence, a
nucleotide sequence encoding a Cas12J polypeptide (e.g., a wild
type Cas12J protein, a nickase Cas12J protein, a dCas12J protein,
fusion Cas12J protein, and the like), a Cas12J guide RNA, and a
nucleotide sequence encoding a Cas12J guide RNA. The present
disclosure provides a nucleic acid comprising a nucleotide sequence
encoding a Cas12J fusion polypeptide. The present disclosure
provides a recombinant expression vector that comprises a
nucleotide sequence encoding a Cas12J polypeptide. The present
disclosure provides a recombinant expression vector that comprises
a nucleotide sequence encoding a Cas12J fusion polypeptide. The
present disclosure provides a recombinant expression vector that
comprises: a) a nucleotide sequence encoding a Cas12J polypeptide;
and b) a nucleotide sequence encoding a Cas12J guide RNA(s). The
present disclosure provides a recombinant expression vector that
comprises: a) a nucleotide sequence encoding a Cas12J fusion
polypeptide; and b) a nucleotide sequence encoding a Cas12J guide
RNA(s). In some cases, the nucleotide sequence encoding the Cas12J
protein and/or the nucleotide sequence encoding the Cas12J guide
RNA is operably linked to a promoter that is operable in a cell
type of choice (e.g., a prokaryotic cell, a eukaryotic cell, a
plant cell, an animal cell, a mammalian cell, a primate cell, a
rodent cell, a human cell, etc.).
[0245] In some cases, a nucleotide sequence encoding a Cas12J
polypeptide of the present disclosure is codon optimized. This type
of optimization can entail a mutation of a Cas12J--encoding
nucleotide sequence to mimic the codon preferences of the intended
host organism or cell while encoding the same protein. Thus, the
codons can be changed, but the encoded protein remains unchanged.
For example, if the intended target cell was a human cell, a human
codon-optimized Cas12J-encoding nucleotide sequence could be used.
As another non-limiting example, if the intended host cell were a
mouse cell, then a mouse codon-optimized Cas12J-encoding nucleotide
sequence could be generated. As another non-limiting example, if
the intended host cell were a plant cell, then a plant
codon-optimized Cas12J-encoding nucleotide sequence could be
generated. As another non-limiting example, if the intended host
cell were an insect cell, then an insect codon-optimized
Cas12J-encoding nucleotide sequence could be generated.
[0246] Codon usage tables are readily available, for example, at
the "Codon Usage Database" available at
www[dot]kazusa[dot]or[dot]jp[forwardslash]codon. In some cases, a
nucleic acid of the present disclosure comprises a Cas12J
polypeptide-encoding nucleotide sequence that is codon optimized
for expression in a eukaryotic cell. In some cases, a nucleic acid
of the present disclosure comprises a Cas12J polypeptide-encoding
nucleotide sequence that is codon optimized for expression in an
animal cell. In some cases, a nucleic acid of the present
disclosure comprises a Cas12J polypeptide-encoding nucleotide
sequence that is codon optimized for expression in a fungus cell.
In some cases, a nucleic acid of the present disclosure comprises a
Cas12J polypeptide-encoding nucleotide sequence that is codon
optimized for expression in a plant cell. In some cases, a nucleic
acid of the present disclosure comprises a Cas12J
polypeptide-encoding nucleotide sequence that is codon optimized
for expression in a monocotyledonous plant species. In some cases,
a nucleic acid of the present disclosure comprises a Cas12J
polypeptide-encoding nucleotide sequence that is codon optimized
for expression in a dicotyledonous plant species. In some cases, a
nucleic acid of the present disclosure comprises a Cas12J
polypeptide-encoding nucleotide sequence that is codon optimized
for expression in a gymnosperm plant species. In some cases, a
nucleic acid of the present disclosure comprises a Cas12J
polypeptide-encoding nucleotide sequence that is codon optimized
for expression in an angiosperm plant species. In some cases, a
nucleic acid of the present disclosure comprises a Cas12J
polypeptide-encoding nucleotide sequence that is codon optimized
for expression in a corn cell. In some cases, a nucleic acid of the
present disclosure comprises a Cas12J polypeptide-encoding
nucleotide sequence that is codon optimized for expression in a
soybean cell. In some cases, a nucleic acid of the present
disclosure comprises a Cas12J polypeptide-encoding nucleotide
sequence that is codon optimized for expression in a rice cell. In
some cases, a nucleic acid of the present disclosure comprises a
Cas12J polypeptide-encoding nucleotide sequence that is codon
optimized for expression in a wheat cell. In some cases, a nucleic
acid of the present disclosure comprises a Cas12J
polypeptide-encoding nucleotide sequence that is codon optimized
for expression in a cotton cell. In some cases, a nucleic acid of
the present disclosure comprises a Cas12J polypeptide-encoding
nucleotide sequence that is codon optimized for expression in a
sorghum cell. In some cases, a nucleic acid of the present
disclosure comprises a Cas12J polypeptide-encoding nucleotide
sequence that is codon optimized for expression in an alfalfa cell.
In some cases, a nucleic acid of the present disclosure comprises a
Cas12J polypeptide-encoding nucleotide sequence that is codon
optimized for expression in a sugar cane cell. In some cases, a
nucleic acid of the present disclosure comprises a Cas12J
polypeptide-encoding nucleotide sequence that is codon optimized
for expression in an Arabidopsis cell. In some cases, a nucleic
acid of the present disclosure comprises a Cas12J
polypeptide-encoding nucleotide sequence that is codon optimized
for expression in a tomato cell. In some cases, a nucleic acid of
the present disclosure comprises a Cas12J polypeptide-encoding
nucleotide sequence that is codon optimized for expression in a
cucumber cell. In some cases, a nucleic acid of the present
disclosure comprises a Cas12J polypeptide-encoding nucleotide
sequence that is codon optimized for expression in a potato cell.
In some cases, a nucleic acid of the present disclosure comprises a
Cas12J polypeptide-encoding nucleotide sequence that is codon
optimized for expression in an algae cell.
[0247] The present disclosure provides one or more recombinant
expression vectors that include (in different recombinant
expression vectors in some cases, and in the same recombinant
expression vector in some cases): (i) a nucleotide sequence of a
donor template nucleic acid (where the donor template comprises a
nucleotide sequence having homology to a target sequence of a
target nucleic acid (e.g., a target genome)); (ii) a nucleotide
sequence that encodes a Cas12J guide RNA that hybridizes to a
target sequence of the target locus of the targeted genome (e.g.,
operably linked to a promoter that is operable in a target cell
such as a eukaryotic cell); and (iii) a nucleotide sequence
encoding a Cas12J protein (e.g., operably linked to a promoter that
is operable in a target cell such as a eukaryotic cell). The
present disclosure provides one or more recombinant expression
vectors that include (in different recombinant expression vectors
in some cases, and in the same recombinant expression vector in
some cases): (i) a nucleotide sequence of a donor template nucleic
acid (where the donor template comprises a nucleotide sequence
having homology to a target sequence of a target nucleic acid
(e.g., a target genome)); and (ii) a nucleotide sequence that
encodes a Cas12J guide RNA that hybridizes to a target sequence of
the target locus of the targeted genome (e.g., operably linked to a
promoter that is operable in a target cell such as a eukaryotic
cell). The present disclosure provides one or more recombinant
expression vectors that include (in different recombinant
expression vectors in some cases, and in the same recombinant
expression vector in some cases): (i) a nucleotide sequence that
encodes a Cas12J guide RNA that hybridizes to a target sequence of
the target locus of the targeted genome (e.g., operably linked to a
promoter that is operable in a target cell such as a eukaryotic
cell); and (ii) a nucleotide sequence encoding a Cas12J protein
(e.g., operably linked to a promoter that is operable in a target
cell such as a eukaryotic cell).
[0248] Suitable expression vectors include viral expression vectors
(e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus
(see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994;
Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS
92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999;
WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and
WO 95/00655); adeno-associated virus (AAV) (see, e.g., Ali et al.,
Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921,
1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997;
Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene
Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996;
Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989)
63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and
Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex
virus; human immunodeficiency virus (see, e.g., Miyoshi et al.,
PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816,
1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen
necrosis virus, and vectors derived from retroviruses such as Rous
Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a
lentivirus, human immunodeficiency virus, myeloproliferative
sarcoma virus, and mammary tumor virus); and the like. In some
cases, a recombinant expression vector of the present disclosure is
a recombinant adeno-associated virus (AAV) vector. In some cases, a
recombinant expression vector of the present disclosure is a
recombinant lentivirus vector. In some cases, a recombinant
expression vector of the present disclosure is a recombinant
retroviral vector.
[0249] For plant applications, viral vectors based on
Tobamoviruses, Potexviruses, Potyviruses, Tobraviruses,
Tombusviruses, Geminiviruses, Bromoviruses, Carmoviruses,
Alfamoviruses, or Cucumoviruses can be used. See, e.g., Peyret and
Lomonossoff (2015) Plant Biotechnol. J. 13:1121. Suitable
Tobamovirus vectors include, for example, a tomato mosaic virus
(ToMV) vector, a tobacco mosaic virus (TMV) vector, a tobacco mild
green mosaic virus (TMGMV) vector, a pepper mild mottle virus
(PMMoV) vector, a paprika mild mottle virus (PaMMV) vector, a
cucumber green mottle mosaic virus (CGMMV) vector, a kyuri green
mottle mosaic virus (KGMMV) vector, a hibiscus latent fort pierce
virus (HLFPV) vector, an odontoglossum ringspot virus (ORSV)
vector, a rehmannia mosaic virus (ReMV) vector, a Sammon's opuntia
virus (SOV) vector, a wasabi mottle virus (WMoV) vector, a youcai
mosaic virus (YoMV) vector, a sunn-hemp mosaic virus (SHMV) vector,
and the like. Suitable Potexvirus vectors include, for example, a
potato virus X (PVX) vector, a potato aucubamosaicvirus (PAMV)
vector, an Alstroemeria virus X (AlsVX) vector, a cactus virus X
(CVX) vector, a Cymbidium mosaic virus (CymMV) vector, a hosta
virus X (HVX) vector, a lily virus X (LVX) vector, a Narcissus
mosaic virus (NMV) vector, a Nerine virus X (NVX) vector, a
Plantago asiatica mosaic virus (PlAMV) vector, a strawberry mild
yellow edge virus (SMYEV) vector, a tulip virus X (TVX) vector, a
white clover mosaic virus (WClMV) vector, a bamboo mosaic virus
(BaMV) vector, and the like. Suitable Potyvirus vectors include,
for example, a potato virus Y (PVY) vector, a bean common mosaic
virus (BCMV) vector, a clover yellow vein virus (ClYVV) vector, an
East Asian Passiflora virus (EAPV) vector, a Freesia mosaic virus
(FreMV) vector, a Japanese yam mosaic virus (JYMV) vector, a
lettuce mosaic virus (LMV) vector, a Maize dwarf mosaic virus
(MDMV) vector, an onion yellow dwarf virus (OYDV) vector, a papaya
ringspot virus (PRSV) vector, a pepper mottle virus (PepMoV)
vector, a Perilla mottle virus (PerMoV) vector, a plum pox virus
(PPV) vector, a potato virus A (PVA) vector, a sorghum mosaic virus
(SrMV) vector, a soybean mosaic virus (SMV) vector, a sugarcane
mosaic virus (SCMV) vector, a tulip mosaic virus (TuIMV) vector, a
turnip mosaic virus (TuMV) vector, a watermelon mosaic virus (WMV)
vector, a zucchini yellow mosaic virus (ZYMV) vector, a tobacco
etch virus (TEV) vector, and the like. Suitable Tobravirus vectors
include, for example, a tobacco rattle virus (TRV) vector and the
like. Suitable Tombusvirus vectors include, for example, a tomato
bushy stunt virus (TBSV) vector, an eggplant mottled crinkle virus
(EMCV) vector, a grapevine Algerian latent virus (GALV) vector, and
the like. Suitable Cucumovirus vectors include, for example, a
cucumber mosaic virus (CMV) vector, a peanut stunt virus (PSV)
vector, a tomato aspermy virus (TAV) vector, and the like. Suitable
Bromovirus vectors include, for example, a brome mosaic virus (BMV)
vector, a cowpea chlorotic mottle virus (CCMV) vector, and the
like. Suitable Carmovirus vectors include, for example, a carnation
mottle virus (CarMV) vector, a melon necrotic spot virus (MNSV)
vector, a pea stem necrotic virus (PSNV) vector, a turnip crinkle
virus (TCV) vector, and the like. Suitable Alfamovirus vectors
include, for example, an alfalfa mosaic virus (AMV) vector, and the
like.
[0250] Depending on the host/vector system utilized, any of a
number of suitable transcription and translation control elements,
including constitutive and inducible promoters, transcription
enhancer elements, transcription terminators, etc. may be used in
the expression vector.
[0251] In some embodiments, a nucleotide sequence encoding a Cas12J
guide RNA is operably linked to a control element, e.g., a
transcriptional control element, such as a promoter. In some
embodiments, a nucleotide sequence encoding a Cas12J protein or a
Cas12J fusion polypeptide is operably linked to a control element,
e.g., a transcriptional control element, such as a promoter.
[0252] The transcriptional control element can be a promoter. In
some cases, the promoter is a constitutively active promoter. In
some cases, the promoter is a regulatable promoter. In some cases,
the promoter is an inducible promoter. In some cases, the promoter
is a tissue-specific promoter. In some cases, the promoter is a
cell type-specific promoter. In some cases, the transcriptional
control element (e.g., the promoter) is functional in a targeted
cell type or targeted cell population. For example, in some cases,
the transcriptional control element can be functional in eukaryotic
cells, e.g., hematopoietic stem cells (e.g., mobilized peripheral
blood (mPB) CD34(+) cell, bone marrow (BM) CD34(+) cell, etc.).
[0253] Non-limiting examples of eukaryotic promoters (promoters
functional in a eukaryotic cell) include EF1.alpha., those from
cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV)
thymidine kinase, early and late SV40, long terminal repeats (LTRs)
from retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art. The expression vector may also contain a
ribosome binding site for translation initiation and a
transcription terminator. The expression vector may also include
appropriate sequences for amplifying expression. The expression
vector may also include nucleotide sequences encoding protein tags
(e.g., 6.times.His tag, hemagglutinin tag, fluorescent protein,
etc.) that can be fused to the Cas12J protein, thus resulting in a
fusion Cas12J polypeptide.
[0254] In some embodiments, a nucleotide sequence encoding a Cas12J
guide RNA and/or a Cas12J fusion polypeptide is operably linked to
an inducible promoter. In some embodiments, a nucleotide sequence
encoding a Cas12J guide RNA and/or a Cas12J fusion protein is
operably linked to a constitutive promoter.
[0255] A promoter can be a constitutively active promoter (i.e., a
promoter that is constitutively in an active/"ON" state), it may be
an inducible promoter (i.e., a promoter whose state, active/"ON" or
inactive/"OFF", is controlled by an external stimulus, e.g., the
presence of a particular temperature, compound, or protein.), it
may be a spatially restricted promoter (i.e., transcriptional
control element, enhancer, etc.)(e.g., tissue specific promoter,
cell type specific promoter, etc.), and it may be a temporally
restricted promoter (i.e., the promoter is in the "ON" state or
"OFF" state during specific stages of embryonic development or
during specific stages of a biological process, e.g., hair follicle
cycle in mice).
[0256] Suitable promoters can be derived from viruses and can
therefore be referred to as viral promoters, or they can be derived
from any organism, including prokaryotic or eukaryotic organisms.
Suitable promoters can be used to drive expression by any RNA
polymerase (e.g., pol I, pol II, pol III). Exemplary promoters
include, but are not limited to the SV40 early promoter, mouse
mammary tumor virus long terminal repeat (LTR) promoter; adenovirus
major late promoter (Ad MLP); a herpes simplex virus (HSV)
promoter, a cytomegalovirus (CMV) promoter such as the CMV
immediate early promoter region (CMVIE), a rous sarcoma virus (RSV)
promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al.,
Nature Biotechnology 20, 497-500 (2002)), an enhanced U6 promoter
(e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), a human
H1 promoter (H1), and the like.
[0257] In some cases, a nucleotide sequence encoding a Cas12J guide
RNA is operably linked to (under the control of) a promoter
operable in a eukaryotic cell (e.g., a U6 promoter, an enhanced U6
promoter, an H1 promoter, and the like). As would be understood by
one of ordinary skill in the art, when expressing an RNA (e.g., a
guide RNA) from a nucleic acid (e.g., an expression vector) using a
U6 promoter (e.g., in a eukaryotic cell), or another PolIII
promoter, the RNA may need to be mutated if there are several Ts in
a row (coding for Us in the RNA). This is because a string of Ts
(e.g., 5 Ts) in DNA can act as a terminator for polymerase III
(PolIII). Thus, in order to ensure transcription of a guide RNA in
a eukaryotic cell it may sometimes be necessary to modify the
sequence encoding the guide RNA to eliminate runs of Ts. In some
cases, a nucleotide sequence encoding a Cas12J protein (e.g., a
wild type Cas12J protein, a nickase Cas12J protein, a dCas12J
protein, a fusion Cas12J protein and the like) is operably linked
to a promoter operable in a eukaryotic cell (e.g., a CMV promoter,
an EF1.alpha. promoter, an estrogen receptor-regulated promoter,
and the like).
[0258] Examples of inducible promoters include, but are not limited
to T7 RNA polymerase promoter, T3 RNA polymerase promoter,
Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter,
lactose induced promoter, heat shock promoter,
Tetracycline-regulated promoter, Steroid-regulated promoter,
Metal-regulated promoter, estrogen receptor-regulated promoter,
etc. Inducible promoters can therefore be regulated by molecules
including, but not limited to, doxycycline; estrogen and/or an
estrogen analog; IPTG; etc.
[0259] Inducible promoters suitable for use include any inducible
promoter described herein or known to one of ordinary skill in the
art. Examples of inducible promoters include, without limitation,
chemically/biochemically-regulated and physically-regulated
promoters such as alcohol-regulated promoters,
tetracycline-regulated promoters (e.g., anhydrotetracycline
(aTc)-responsive promoters and other tetracycline-responsive
promoter systems, which include a tetracycline repressor protein
(tetR), a tetracycline operator sequence (tetO) and a tetracycline
transactivator fusion protein (tTA)), steroid-regulated promoters
(e.g., promoters based on the rat glucocorticoid receptor, human
estrogen receptor, moth ecdysone receptors, and promoters from the
steroid/retinoid/thyroid receptor superfamily), metal-regulated
promoters (e.g., promoters derived from metallothionein (proteins
that bind and sequester metal ions) genes from yeast, mouse and
human), pathogenesis-regulated promoters (e.g., induced by
salicylic acid, ethylene or benzothiadiazole (BTH)),
temperature/heat-inducible promoters (e.g., heat shock promoters),
and light-regulated promoters (e.g., light responsive promoters
from plant cells).
[0260] In some cases, the promoter is a spatially restricted
promoter (i.e., cell type specific promoter, tissue specific
promoter, etc.) such that in a multi-cellular organism, the
promoter is active (i.e., "ON") in a subset of specific cells.
Spatially restricted promoters may also be referred to as
enhancers, transcriptional control elements, control sequences,
etc. Any convenient spatially restricted promoter may be used as
long as the promoter is functional in the targeted host cell (e.g.,
eukaryotic cell; prokaryotic cell).
[0261] In some cases, the promoter is a reversible promoter.
Suitable reversible promoters, including reversible inducible
promoters are known in the art. Such reversible promoters may be
isolated and derived from many organisms, e.g., eukaryotes and
prokaryotes. Modification of reversible promoters derived from a
first organism for use in a second organism, e.g., a first
prokaryote and a second a eukaryote, a first eukaryote and a second
a prokaryote, etc., is well known in the art. Such reversible
promoters, and systems based on such reversible promoters but also
comprising additional control proteins, include, but are not
limited to, alcohol regulated promoters (e.g., alcohol
dehydrogenase I (alcA) gene promoter, promoters responsive to
alcohol transactivator proteins (AlcR), etc.), tetracycline
regulated promoters, (e.g., promoter systems including
TetActivators, TetON, TetOFF, etc.), steroid regulated promoters
(e.g., rat glucocorticoid receptor promoter systems, human estrogen
receptor promoter systems, retinoid promoter systems, thyroid
promoter systems, ecdysone promoter systems, mifepristone promoter
systems, etc.), metal regulated promoters (e.g., metallothionein
promoter systems, etc.), pathogenesis-related regulated promoters
(e.g., salicylic acid regulated promoters, ethylene regulated
promoters, benzothiadiazole regulated promoters, etc.), temperature
regulated promoters (e.g., heat shock inducible promoters (e.g.,
HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated
promoters, synthetic inducible promoters, and the like.
[0262] RNA polymerase III (Pol III) promoters can be used to drive
the expression of non-protein coding RNA molecules (e.g., guide
RNAs). In some cases, a suitable promoter is a Pol III promoter. In
some cases, a Pol III promoter is operably linked to a nucleotide
sequence encoding a guide RNA (gRNA). In some cases, a Pol III
promoter is operably linked to a nucleotide sequence encoding a
single-guide RNA (sgRNA). In some cases, a Pol III promoter is
operably linked to a nucleotide sequence encoding a CRISPR RNA
(crRNA). In some cases, a Pol III promoter is operably linked to a
nucleotide sequence encoding a encoding a tracrRNA.
[0263] Non-limiting examples of Pol III promoters include a U6
promoter, an Hl promoter, a 5S promoter, an Adenovirus 2 (Ad2) VAI
promoter, a tRNA promoter, and a 7SK promoter. See, for example,
Schramm and Hernandez (2002) Genes & Development 16:2593-2620.
In some cases, a Pol III promoter is selected from the group
consisting of a U6 promoter, an Hl promoter, a 5S promoter, an
Adenovirus 2 (Ad2) VAI promoter, a tRNA promoter, and a 7SK
promoter. In some cases, a guide RNA-encoding nucleotide sequence
is operably linked to a promoter selected from the group consisting
of a U6 promoter, an Hl promoter, a 5S promoter, an Adenovirus 2
(Ad2) VAI promoter, a tRNA promoter, and a 7SK promoter. In some
cases, a single-guide RNA-encoding nucleotide sequence is operably
linked to a promoter selected from the group consisting of a U6
promoter, an Hl promoter, a 5S promoter, an Adenovirus 2 (Ad2) VAI
promoter, a tRNA promoter, and a 7SK promoter.
[0264] Examples describing a promoter that can be used herein in
connection with expression in plants, plant tissues, and plant
cells include, but are not limited to, promoters described in: U.S.
Pat. No. 6,437,217 (maize RS81 promoter), U.S. Pat. No. 5,641,876
(rice actin promoter), U.S. Pat. No. 6,426,446 (maize RS324
promoter), U.S. Pat. No. 6,429,362 (maize PR-1 promoter), U.S. Pat.
No. 6,232,526 (maize A3 promoter), U.S. Pat. No. 6,177,611
(constitutive maize promoters), U.S. Pat. Nos. 5,322,938,
5,352,605, 5,359,142 and 5,530,196 (35S promoter), U.S. Pat. No.
6,433,252 (maize L3 oleosin promoter), U.S. Pat. No. 6,429,357
(rice actin 2 promoter as well as a rice actin 2 intron), U.S. Pat.
No. 5,837,848 (root specific promoter), U.S. Pat. No. 6,294,714
(light inducible promoters), U.S. Pat. No. 6,140,078 (salt
inducible promoters), U.S. Pat. No. 6,252,138 (pathogen inducible
promoters), U.S. Pat. No. 6,175,060 (phosphorus deficiency
inducible promoters), U.S. Pat. No. 6,635,806 (gamma-coixin
promoter), and U.S. patent application Ser. No. 09/757,089 (maize
chloroplast aldolase promoter). Additional promoters that can find
use include a nopaline synthase (NOS) promoter (Ebert et al.,
1987), the octopine synthase (OCS) promoter (which is carried on
tumor-inducing plasmids of Agrobacterium tumefaciens), the
caulimovirus promoters such as the cauliflower mosaic virus (CaMV)
19S promoter (Lawton et al. Plant Molecular Biology (1987) 9:
315-324), the CaMV 35S promoter (Odell et al., Nature (1985) 313:
810-812), the figwort mosaic virus 35S-promoter (U.S. Pat. Nos.
6,051,753; 5,378,619), the sucrose synthase promoter (Yang and
Russell, Proceedings of the National Academy of Sciences, USA
(1990) 87: 4144-4148), the R gene complex promoter (Chandler et
al., Plant Cell (1989) 1: 1175-1183), and the chlorophyll a/b
binding protein gene promoter, PH1SV (U.S. Pat. No. 5,850,019), and
AGRtu.nos (GenBank Accession V00087; Depicker et al., Journal of
Molecular and Applied Genetics (1982) 1: 561-573; Bevan et al.,
1983) promoters.
[0265] Methods of introducing a nucleic acid (e.g., a nucleic acid
comprising a donor polynucleotide sequence, one or more nucleic
acids encoding a Cas12J protein and/or a Cas12J guide RNA, and the
like) into a host cell are known in the art, and any convenient
method can be used to introduce a nucleic acid (e.g., an expression
construct) into a cell. Suitable methods include e.g., viral
infection, transfection, lipofection, electroporation, calcium
phosphate precipitation, polyethyleneimine (PEI)-mediated
transfection, DEAE-dextran mediated transfection, liposome-mediated
transfection, particle gun technology, calcium phosphate
precipitation, direct microinjection, nanoparticle-mediated nucleic
acid delivery, and the like.
[0266] Introducing the recombinant expression vector into cells can
occur in any culture media and under any culture conditions that
promote the survival of the cells. Introducing the recombinant
expression vector into a target cell can be carried out in vivo or
ex vivo. Introducing the recombinant expression vector into a
target cell can be carried out in vitro.
[0267] In some embodiments, a Cas12J protein can be provided as
RNA. The RNA can be provided by direct chemical synthesis or may be
transcribed in vitro from a DNA (e.g., encoding the Cas12J
protein). Once synthesized, the RNA may be introduced into a cell
by any of the well-known techniques for introducing nucleic acids
into cells (e.g., microinjection, electroporation, transfection,
etc.).
[0268] Nucleic acids may be provided to the cells using
well-developed transfection techniques; see, e.g. Angel and Yanik
(2010) PLoS ONE 5(7): e11756, and the commercially available
TransMessenger.RTM. reagents from Qiagen, Stemfect.TM. RNA
Transfection Kit from Stemgent, and TransIT.RTM.-mRNA Transfection
Kit from Mirus Bio LLC. See also Beumer et al. (2008) PNAS
105(50):19821-19826.
[0269] Vectors may be provided directly to a target host cell. In
other words, the cells are contacted with vectors comprising the
subject nucleic acids (e.g., recombinant expression vectors having
the donor template sequence and encoding the Cas12J guide RNA;
recombinant expression vectors encoding the Cas12J protein; etc.)
such that the vectors are taken up by the cells. Methods for
contacting cells with nucleic acid vectors that are plasmids,
include electroporation, calcium chloride transfection,
microinjection, and lipofection are well known in the art. For
viral vector delivery, cells can be contacted with viral particles
comprising the subject viral expression vectors.
[0270] Retroviruses, for example, lentiviruses, are suitable for
use in methods of the present disclosure. Commonly used retroviral
vectors are "defective", i.e. unable to produce viral proteins
required for productive infection. Rather, replication of the
vector requires growth in a packaging cell line. To generate viral
particles comprising nucleic acids of interest, the retroviral
nucleic acids comprising the nucleic acid are packaged into viral
capsids by a packaging cell line. Different packaging cell lines
provide a different envelope protein (ecotropic, amphotropic or
xenotropic) to be incorporated into the capsid, this envelope
protein determining the specificity of the viral particle for the
cells (ecotropic for murine and rat; amphotropic for most mammalian
cell types including human, dog and mouse; and xenotropic for most
mammalian cell types except murine cells). The appropriate
packaging cell line may be used to ensure that the cells are
targeted by the packaged viral particles. Methods of introducing
subject vector expression vectors into packaging cell lines and of
collecting the viral particles that are generated by the packaging
lines are well known in the art. Nucleic acids can also introduced
by direct micro-injection (e.g., injection of RNA).
[0271] Vectors used for providing the nucleic acids encoding Cas12J
guide RNA and/or a Cas12J polypeptide to a target host cell can
include suitable promoters for driving the expression, that is,
transcriptional activation, of the nucleic acid of interest. In
other words, in some cases, the nucleic acid of interest will be
operably linked to a promoter. This may include ubiquitously acting
promoters, for example, the CMV-.beta.-actin promoter, or inducible
promoters, such as promoters that are active in particular cell
populations or that respond to the presence of drugs such as
tetracycline. By transcriptional activation, it is intended that
transcription will be increased above basal levels in the target
cell by 10 fold, by 100 fold, more usually by 1000 fold. In
addition, vectors used for providing a nucleic acid encoding a
Cas12J guide RNA and/or a Cas12J protein to a cell may include
nucleic acid sequences that encode for selectable markers in the
target cells, so as to identify cells that have taken up the Cas12J
guide RNA and/or Cas12J protein.
[0272] A nucleic acid comprising a nucleotide sequence encoding a
Cas12J polypeptide, or a Cas12J fusion polypeptide, is in some
cases an RNA. Thus, a Cas12J fusion protein can be introduced into
cells as RNA. Methods of introducing RNA into cells are known in
the art and may include, for example, direct injection,
transfection, or any other method used for the introduction of DNA.
A Cas12J protein may instead be provided to cells as a polypeptide.
Such a polypeptide may optionally be fused to a polypeptide domain
that increases solubility of the product. The domain may be linked
to the polypeptide through a defined protease cleavage site, e.g. a
TEV sequence, which is cleaved by TEV protease. The linker may also
include one or more flexible sequences, e.g. from 1 to 10 glycine
residues. In some embodiments, the cleavage of the fusion protein
is performed in a buffer that maintains solubility of the product,
e.g. in the presence of from 0.5 to 2 M urea, in the presence of
polypeptides and/or polynucleotides that increase solubility, and
the like. Domains of interest include endosomolytic domains, e.g.
influenza HA domain; and other polypeptides that aid in production,
e.g. IF2 domain, GST domain, GRPE domain, and the like. The
polypeptide may be formulated for improved stability. For example,
the peptides may be PEGylated, where the polyethyleneoxy group
provides for enhanced lifetime in the blood stream.
[0273] Additionally or alternatively, a Cas12J polypeptide of the
present disclosure may be fused to a polypeptide permeant domain to
promote uptake by the cell. A number of permeant domains are known
in the art and may be used in the non-integrating polypeptides of
the present disclosure, including peptides, peptidomimetics, and
non-peptide carriers. For example, a permeant peptide may be
derived from the third alpha helix of Drosophila melanogaster
transcription factor Antennapaedia, referred to as penetratin,
which comprises the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID
NO: 68). As another example, the permeant peptide comprises the
HIV-1 tat basic region amino acid sequence, which may include, for
example, amino acids 49-57 of naturally-occurring tat protein.
Other permeant domains include poly-arginine motifs, for example,
the region of amino acids 34-56 of HIV-1 rev protein,
nona-arginine, octa-arginine, and the like. (See, for example,
Futaki et al. (2003) Curr Protein Pept Sci. 2003 April; 4(2): 87-9
and 446; and Wender et al. (2000) Proc. Natl. Acad. Sci. U.S.A.
2000 Nov. 21; 97(24):13003-8; published U.S. Patent applications
20030220334; 20030083256; 20030032593; and 20030022831, herein
specifically incorporated by reference for the teachings of
translocation peptides and peptoids). The nona-arginine (R9)
sequence is one of the more efficient PTDs that have been
characterized (Wender et al. 2000; Uemura et al. 2002). The site at
which the fusion is made may be selected in order to optimize the
biological activity, secretion or binding characteristics of the
polypeptide. The optimal site will be determined by routine
experimentation.
[0274] As noted above, in some cases, the target cell is a plant
cell. Numerous methods for transforming chromosomes or plastids in
a plant cell with a recombinant nucleic acid are known in the art,
which can be used according to methods of the present application
to produce a transgenic plant cell and/or a transgenic plant. Any
suitable method or technique for transformation of a plant cell
known in the art can be used. Effective methods for transformation
of plants include bacterially mediated transformation, such as
Agrobacterium-mediated or Rhizobium-mediated transformation and
microprojectile bombardment-mediated transformation. A variety of
methods are known in the art for transforming explants with a
transformation vector via bacterially mediated transformation or
microprojectile bombardment and then subsequently culturing, etc.,
those explants to regenerate or develop transgenic plants. Other
methods for plant transformation, such as microinjection,
electroporation, vacuum infiltration, pressure, sonication, silicon
carbide fiber agitation, PEG-mediated transformation, etc., are
also known in the art. Transgenic plants produced by these
transformation methods can be chimeric or non-chimeric for the
transformation event depending on the methods and explants
used.
[0275] Methods of transforming plant cells are well known by
persons of ordinary skill in the art. For instance, specific
instructions for transforming plant cells by microprojectile
bombardment with particles coated with recombinant DNA (e.g.,
biolistic transformation) are found in U.S. Pat. Nos. 5,550,318;
5,538,880 6,160,208; 6,399,861; and 6,153,812 and
Agrobacterium-mediated transformation is described in U.S. Pat.
Nos. 5,159,135; 5,824,877; 5,591,616; 6,384,301; 5,750,871;
5,463,174; and 5,188,958. Additional methods for transforming
plants can be found in, for example, Compendium of Transgenic Crop
Plants (2009) Blackwell Publishing. Any appropriate method known to
those skilled in the art can be used to transform a plant cell with
any of the nucleic acids provided herein.
[0276] A Cas12J polypeptide of the present disclosure may be
produced in vitro or by eukaryotic cells or by prokaryotic cells,
and it may be further processed by unfolding, e.g. heat
denaturation, dithiothreitol reduction, etc. and may be further
refolded, using methods known in the art.
[0277] Modifications of interest that do not alter primary sequence
include chemical derivatization of polypeptides, e.g., acylation,
acetylation, carboxylation, amidation, etc. Also included are
modifications of glycosylation, e.g. those made by modifying the
glycosylation patterns of a polypeptide during its synthesis and
processing or in further processing steps; e.g. by exposing the
polypeptide to enzymes which affect glycosylation, such as
mammalian glycosylating or deglycosylating enzymes. Also embraced
are sequences that have phosphorylated amino acid residues, e.g.
phosphotyrosine, phosphoserine, or phosphothreonine.
[0278] Also suitable for inclusion in embodiments of the present
disclosure are nucleic acids (e.g., encoding a Cas12J guide RNA,
encoding a Cas12J fusion protein, etc.) and proteins (e.g., a
Cas12J fusion protein derived from a wild type protein or a variant
protein) that have been modified using ordinary molecular
biological techniques and synthetic chemistry so as to improve
their resistance to proteolytic degradation, to change the target
sequence specificity, to optimize solubility properties, to alter
protein activity (e.g., transcription modulatory activity,
enzymatic activity, etc.) or to render them more suitable. Analogs
of such polypeptides include those containing residues other than
naturally occurring L-amino acids, e.g. D-amino acids or
non-naturally occurring synthetic amino acids. D-amino acids may be
substituted for some or all of the amino acid residues.
[0279] A Cas12J polypeptide of the present disclosure may be
prepared by in vitro synthesis, using conventional methods as known
in the art. Various commercial synthetic apparatuses are available,
for example, automated synthesizers by Applied Biosystems, Inc.,
Beckman, etc. By using synthesizers, naturally occurring amino
acids may be substituted with unnatural amino acids. The particular
sequence and the manner of preparation will be determined by
convenience, economics, purity required, and the like.
[0280] If desired, various groups may be introduced into the
peptide during synthesis or during expression, which allow for
linking to other molecules or to a surface. Thus, e.g., cysteines
can be used to make thioethers, histidines for linking to a metal
ion complex, carboxyl groups for forming amides or esters, amino
groups for forming amides, and the like.
[0281] A Cas12J polypeptide of the present disclosure may also be
isolated and purified in accordance with conventional methods of
recombinant synthesis. A lysate may be prepared of the expression
host and the lysate purified using high performance liquid
chromatography (HPLC), exclusion chromatography, gel
electrophoresis, affinity chromatography, or other purification
technique. For the most part, the compositions which are used will
comprise 20% or more by weight of the desired product, more usually
75% or more by weight, preferably 95% or more by weight, and for
therapeutic purposes, usually 99.5% or more by weight, in relation
to contaminants related to the method of preparation of the product
and its purification. Usually, the percentages will be based upon
total protein. Thus, in some cases, a Cas12J polypeptide, or a
Cas12J fusion polypeptide, of the present disclosure is at least
80% pure, at least 85% pure, at least 90% pure, at least 95% pure,
at least 98% pure, or at least 99% pure (e.g., free of
contaminants, non-Cas12J proteins or other macromolecules,
etc.).
[0282] To induce cleavage or any desired modification to a target
nucleic acid (e.g., genomic DNA), or any desired modification to a
polypeptide associated with target nucleic acid, the Cas12J guide
RNA and/or the Cas12J polypeptide of the present disclosure and/or
the donor template sequence, whether they be introduced as nucleic
acids or polypeptides, are provided to the cells for about 30
minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5
hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8
hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period
from about 30 minutes to about 24 hours, which may be repeated with
a frequency of about every day to about every 4 days, e.g., every
1.5 days, every 2 days, every 3 days, or any other frequency from
about every day to about every four days. The agent(s) may be
provided to the subject cells one or more times, e.g. one time,
twice, three times, or more than three times, and the cells allowed
to incubate with the agent(s) for some amount of time following
each contacting event e.g. 16-24 hours, after which time the media
is replaced with fresh media and the cells are cultured
further.
[0283] In cases in which two or more different targeting complexes
are provided to the cell (e.g., two different Cas12J guide RNAs
that are complementary to different sequences within the same or
different target nucleic acid), the complexes may be provided
simultaneously (e.g. as two polypeptides and/or nucleic acids), or
delivered simultaneously. Alternatively, they may be provided
consecutively, e.g. the targeting complex being provided first,
followed by the second targeting complex, etc. or vice versa.
[0284] To improve the delivery of a DNA vector into a target cell,
the DNA can be protected from damage and its entry into the cell
facilitated, for example, by using lipoplexes and polyplexes. Thus,
in some cases, a nucleic acid of the present disclosure (e.g., a
recombinant expression vector of the present disclosure) can be
covered with lipids in an organized structure like a micelle or a
liposome. When the organized structure is complexed with DNA it is
called a lipoplex. There are three types of lipids, anionic
(negatively-charged), neutral, or cationic (positively-charged).
Lipoplexes that utilize cationic lipids have proven utility for
gene transfer. Cationic lipids, due to their positive charge,
naturally complex with the negatively charged DNA. Also, as a
result of their charge, they interact with the cell membrane.
Endocytosis of the lipoplex then occurs, and the DNA is released
into the cytoplasm. The cationic lipids also protect against
degradation of the DNA by the cell.
[0285] Complexes of polymers with DNA are called polyplexes. Most
polyplexes consist of cationic polymers and their production is
regulated by ionic interactions. One large difference between the
methods of action of polyplexes and lipoplexes is that polyplexes
cannot release their DNA load into the cytoplasm, so to this end,
co-transfection with endosome-lytic agents (to lyse the endosome
that is made during endocytosis) such as inactivated adenovirus
must occur. However, this is not always the case; polymers such as
polyethylenimine have their own method of endosome disruption as
does chitosan and trimethylchitosan.
[0286] Dendrimers, a highly branched macromolecule with a spherical
shape, may be also be used to genetically modify stem cells. The
surface of the dendrimer particle may be functionalized to alter
its properties. In particular, it is possible to construct a
cationic dendrimer (i.e., one with a positive surface charge). When
in the presence of genetic material such as a DNA plasmid, charge
complementarity leads to a temporary association of the nucleic
acid with the cationic dendrimer. On reaching its destination, the
dendrimer-nucleic acid complex can be taken up into a cell by
endocytosis.
[0287] In some cases, a nucleic acid of the disclosure (e.g., an
expression vector) includes an insertion site for a guide sequence
of interest. For example, a nucleic acid can include an insertion
site for a guide sequence of interest, where the insertion site is
immediately adjacent to a nucleotide sequence encoding the portion
of a Cas12J guide RNA that does not change when the guide sequence
is changed to hybridized to a desired target sequence (e.g.,
sequences that contribute to the Cas12J binding aspect of the guide
RNA, e.g., the sequences that contribute to the dsRNA duplex(es) of
the Cas12J guide RNA--this portion of the guide RNA can also be
referred to as the `scaffold` or `constant region` of the guide
RNA). Thus, in some cases, a subject nucleic acid (e.g., an
expression vector) includes a nucleotide sequence encoding a Cas12J
guide RNA, except that the portion encoding the guide sequence
portion of the guide RNA is an insertion sequence (an insertion
site). An insertion site is any nucleotide sequence used for the
insertion of the desired sequence. "Insertion sites" for use with
various technologies are known to those of ordinary skill in the
art and any convenient insertion site can be used. An insertion
site can be for any method for manipulating nucleic acid sequences.
For example, in some cases the insertion site is a multiple cloning
site (MCS) (e.g., a site including one or more restriction enzyme
recognition sequences), a site for ligation independent cloning, a
site for recombination based cloning (e.g., recombination based on
att sites), a nucleotide sequence recognized by a CRISPR/Cas (e.g.
Cas9) based technology, and the like.
[0288] An insertion site can be any desirable length, and can
depend on the type of insertion site (e.g., can depend on whether
(and how many) the site includes one or more restriction enzyme
recognition sequences, whether the site includes a target site for
a CRISPR/Cas protein, etc.). In some cases, an insertion site of a
subject nucleic acid is 3 or more nucleotides (nt) in length (e.g.,
5 or more, 8 or more, 10 or more, 15 or more, 17 or more, 18 or
more, 19 or more, 20 or more or 25 or more, or 30 or more nt in
length). In some cases, the length of an insertion site of a
subject nucleic acid has a length in a range of from 2 to 50
nucleotides (nt) (e.g., from 2 to 40 nt, from 2 to 30 nt, from 2 to
25 nt, from 2 to 20 nt, from 5 to 50 nt, from 5 to 40 nt, from 5 to
30 nt, from 5 to 25 nt, from 5 to 20 nt, from 10 to 50 nt, from 10
to 40 nt, from 10 to 30 nt, from 10 to 25 nt, from 10 to 20 nt,
from 17 to 50 nt, from 17 to 40 nt, from 17 to 30 nt, from 17 to 25
nt). In some cases, the length of an insertion site of a subject
nucleic acid has a length in a range of from 5 to 40 nt.
Nucleic Acid Modifications
[0289] In some embodiments, a subject nucleic acid (e.g., a Cas12J
guide RNA) has one or more modifications, e.g., a base
modification, a backbone modification, etc., to provide the nucleic
acid with a new or enhanced feature (e.g., improved stability). A
nucleoside is a base-sugar combination. The base portion of the
nucleoside is normally a heterocyclic base. The two most common
classes of such heterocyclic bases are the purines and the
pyrimidines. Nucleotides are nucleosides that further include a
phosphate group covalently linked to the sugar portion of the
nucleoside. For those nucleosides that include a pentofuranosyl
sugar, the phosphate group can be linked to the 2', the 3', or the
5' hydroxyl moiety of the sugar. In forming oligonucleotides, the
phosphate groups covalently link adjacent nucleosides to one
another to form a linear polymeric compound. In turn, the
respective ends of this linear polymeric compound can be further
joined to form a circular compound, however, linear compounds are
suitable. In addition, linear compounds may have internal
nucleotide base complementarity and may therefore fold in a manner
as to produce a fully or partially double-stranded compound. Within
oligonucleotides, the phosphate groups are commonly referred to as
forming the internucleoside backbone of the oligonucleotide. The
normal linkage or backbone of RNA and DNA is a 3' to 5'
phosphodiester linkage.
[0290] Suitable nucleic acid modifications include, but are not
limited to: 2'Omethyl modified nucleotides, 2' Fluoro modified
nucleotides, locked nucleic acid (LNA) modified nucleotides,
peptide nucleic acid (PNA) modified nucleotides, nucleotides with
phosphorothioate linkages, and a 5' cap (e.g., a 7-methylguanylate
cap (m7G)). Additional details and additional modifications are
described below.
[0291] A 2'-O-Methyl modified nucleotide (also referred to as
2'-O-Methyl RNA) is a naturally occurring modification of RNA found
in tRNA and other small RNAs that arises as a post-transcriptional
modification. Oligonucleotides can be directly synthesized that
contain 2'-O-Methyl RNA. This modification increases Tm of RNA:RNA
duplexes but results in only small changes in RNA:DNA stability. It
is stabile with respect to attack by single-stranded ribonucleases
and is typically 5 to 10-fold less susceptible to DNases than DNA.
It is commonly used in antisense oligos as a means to increase
stability and binding affinity to the target message.
[0292] 2' Fluoro modified nucleotides (e.g., 2' Fluoro bases) have
a fluorine modified ribose which increases binding affinity (Tm)
and also confers some relative nuclease resistance when compared to
native RNA. These modifications are commonly employed in ribozymes
and siRNAs to improve stability in serum or other biological
fluids.
[0293] LNA bases have a modification to the ribose backbone that
locks the base in the C3'-endo position, which favors RNA A-type
helix duplex geometry. This modification significantly increases Tm
and is also very nuclease resistant. Multiple LNA insertions can be
placed in an oligo at any position except the 3'-end. Applications
have been described ranging from antisense oligos to hybridization
probes to SNP detection and allele specific PCR. Due to the large
increase in Tm conferred by LNAs, they also can cause an increase
in primer dimer formation as well as self-hairpin formation. In
some cases, the number of LNAs incorporated into a single oligo is
10 bases or less.
[0294] The phosphorothioate (PS) bond (i.e., a phosphorothioate
linkage) substitutes a sulfur atom for a non-bridging oxygen in the
phosphate backbone of a nucleic acid (e.g., an oligo). This
modification renders the internucleotide linkage resistant to
nuclease degradation. Phosphorothioate bonds can be introduced
between the last 3-5 nucleotides at the 5'- or 3'-end of the oligo
to inhibit exonuclease degradation. Including phosphorothioate
bonds within the oligo (e.g., throughout the entire oligo) can help
reduce attack by endonucleases as well.
[0295] In some embodiments, a subject nucleic acid has one or more
nucleotides that are 2'-O-Methyl modified nucleotides. In some
embodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, etc.)
has one or more 2' Fluoro modified nucleotides. In some
embodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, etc.)
has one or more LNA bases. In some embodiments, a subject nucleic
acid (e.g., a dsRNA, a siNA, etc.) has one or more nucleotides that
are linked by a phosphorothioate bond (i.e., the subject nucleic
acid has one or more phosphorothioate linkages). In some
embodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, etc.)
has a 5' cap (e.g., a 7-methylguanylate cap (m7G)). In some
embodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, etc.)
has a combination of modified nucleotides. For example, a subject
nucleic acid (e.g., a dsRNA, a siNA, etc.) can have a 5' cap (e.g.,
a 7-methylguanylate cap (m7G)) in addition to having one or more
nucleotides with other modifications (e.g., a 2'-O-Methyl
nucleotide and/or a 2' Fluoro modified nucleotide and/or a LNA base
and/or a phosphorothioate linkage).
Modified Backbones and Modified Internucleoside Linkages
[0296] Examples of suitable nucleic acids (e.g., a Cas12J guide
RNA) containing modifications include nucleic acids containing
modified backbones or non-natural internucleoside linkages. Nucleic
acids having modified backbones include those that retain a
phosphorus atom in the backbone and those that do not have a
phosphorus atom in the backbone.
[0297] Suitable modified oligonucleotide backbones containing a
phosphorus atom therein include, for example, phosphorothioates,
chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates, 5'-alkylene phosphonates and
chiral phosphonates, phosphinates, phosphoramidates including
3'-amino phosphoramidate and aminoalkylphosphoramidates,
phosphorodiamidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters,
selenophosphates and boranophosphates having normal 3'-5' linkages,
2'-5' linked analogs of these, and those having inverted polarity
wherein one or more internucleotide linkages is a 3' to 3', 5' to
5' or 2' to 2' linkage. Suitable oligonucleotides having inverted
polarity comprise a single 3' to 3' linkage at the 3'-most
internucleotide linkage i.e. a single inverted nucleoside residue
which may be a basic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts (such as, for example,
potassium or sodium), mixed salts and free acid forms are also
included.
[0298] In some embodiments, a subject nucleic acid comprises one or
more phosphorothioate and/or heteroatom internucleoside linkages,
in particular --CH.sub.2--NHO--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-(known as a methylene
(methylimino) or MMI backbone),
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- (wherein the native
phosphodiester internucleotide linkage is represented as
--O--P(.dbd.O)(OH)--O--CH.sub.2--). MMI type internucleoside
linkages are disclosed in the above referenced U.S. Pat. No.
5,489,677, the disclosure of which is incorporated herein by
reference in its entirety. Suitable amide internucleoside linkages
are disclosed in U.S. Pat. No. 5,602,240, the disclosure of which
is incorporated herein by reference in its entirety.
[0299] Also suitable are nucleic acids having morpholino backbone
structures as described in, e.g., U.S. Pat. No. 5,034,506. For
example, in some embodiments, a subject nucleic acid comprises a
6-membered morpholino ring in place of a ribose ring. In some of
these embodiments, a phosphorodiamidate or other non-phosphodiester
internucleoside linkage replaces a phosphodiester linkage.
[0300] Suitable modified polynucleotide backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; riboacetyl backbones; alkene containing backbones;
sulfamate backbones; methyleneimino and methylenehydrazino
backbones; sulfonate and sulfonamide backbones; amide backbones;
and others having mixed N, O, S and CH.sub.2 component parts.
Mimetics
[0301] A subject nucleic acid can be a nucleic acid mimetic. The
term "mimetic" as it is applied to polynucleotides is intended to
include polynucleotides wherein only the furanose ring or both the
furanose ring and the internucleotide linkage are replaced with
non-furanose groups, replacement of only the furanose ring is also
referred to in the art as being a sugar surrogate. The heterocyclic
base moiety or a modified heterocyclic base moiety is maintained
for hybridization with an appropriate target nucleic acid. One such
nucleic acid, a polynucleotide mimetic that has been shown to have
excellent hybridization properties, is referred to as a peptide
nucleic acid (PNA). In PNA, the sugar-backbone of a polynucleotide
is replaced with an amide containing backbone, in particular an
aminoethylglycine backbone. The nucleotides are retained and are
bound directly or indirectly to aza nitrogen atoms of the amide
portion of the backbone.
[0302] One polynucleotide mimetic that has been reported to have
excellent hybridization properties is a peptide nucleic acid (PNA).
The backbone in PNA compounds is two or more linked
aminoethylglycine units which gives PNA an amide containing
backbone. The heterocyclic base moieties are bound directly or
indirectly to aza nitrogen atoms of the amide portion of the
backbone. Representative U.S. patents that describe the preparation
of PNA compounds include, but are not limited to: U.S. Pat. Nos.
5,539,082; 5,714,331; and 5,719,262, the disclosures of which are
incorporated herein by reference in their entirety.
[0303] Another class of polynucleotide mimetic that has been
studied is based on linked morpholino units (morpholino nucleic
acid) having heterocyclic bases attached to the morpholino ring. A
number of linking groups have been reported that link the
morpholino monomeric units in a morpholino nucleic acid. One class
of linking groups has been selected to give a non-ionic oligomeric
compound. The non-ionic morpholino-based oligomeric compounds are
less likely to have undesired interactions with cellular proteins.
Morpholino-based polynucleotides are non-ionic mimics of
oligonucleotides which are less likely to form undesired
interactions with cellular proteins (Dwaine A. Braasch and David R.
Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based
polynucleotides are disclosed in U.S. Pat. No. 5,034,506, the
disclosure of which is incorporated herein by reference in its
entirety. A variety of compounds within the morpholino class of
polynucleotides have been prepared, having a variety of different
linking groups joining the monomeric subunits.
[0304] A further class of polynucleotide mimetic is referred to as
cyclohexenyl nucleic acids (CeNA). The furanose ring normally
present in a DNA/RNA molecule is replaced with a cyclohexenyl ring.
CeNA DMT protected phosphoramidite monomers have been prepared and
used for oligomeric compound synthesis following classical
phosphoramidite chemistry. Fully modified CeNA oligomeric compounds
and oligonucleotides having specific positions modified with CeNA
have been prepared and studied (see Wang et al., J. Am. Chem. Soc.,
2000, 122, 8595-8602, the disclosure of which is incorporated
herein by reference in its entirety). In general the incorporation
of CeNA monomers into a DNA chain increases its stability of a
DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and
DNA complements with similar stability to the native complexes. The
study of incorporating CeNA structures into natural nucleic acid
structures was shown by NMR and circular dichroism to proceed with
easy conformational adaptation.
[0305] A further modification includes Locked Nucleic Acids (LNAs)
in which the 2'-hydroxyl group is linked to the 4' carbon atom of
the sugar ring thereby forming a 2'-C,4'-C-oxymethylene linkage
thereby forming a bicyclic sugar moiety. The linkage can be a
methylene (--CH.sub.2--), group bridging the 2' oxygen atom and the
4' carbon atom wherein n is 1 or 2 (Singh et al., Chem. Commun.,
1998, 4, 455-456, the disclosure of which is incorporated herein by
reference in its entirety). LNA and LNA analogs display very high
duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to
+10.degree. C.), stability towards 3'-exonucleolytic degradation
and good solubility properties. Potent and nontoxic antisense
oligonucleotides containing LNAs have been described (e.g.,
Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97,
5633-5638, the disclosure of which is incorporated herein by
reference in its entirety).
[0306] The synthesis and preparation of the LNA monomers adenine,
cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along
with their oligomerization, and nucleic acid recognition properties
have been described (e.g., Koshkin et al., Tetrahedron, 1998, 54,
3607-3630, the disclosure of which is incorporated herein by
reference in its entirety). LNAs and preparation thereof are also
described in WO 98/39352 and WO 99/14226, as well as U.S.
applications 20120165514, 20100216983, 20090041809, 20060117410,
20040014959, 20020094555, and 20020086998, the disclosures of which
are incorporated herein by reference in their entirety.
Modified Sugar Moieties
[0307] A subject nucleic acid can also include one or more
substituted sugar moieties. Suitable polynucleotides comprise a
sugar substituent group selected from: OH; F; O-, S-, or N-alkyl;
O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl,
wherein the alkyl, alkenyl and alkynyl may be substituted or
unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10
alkenyl and alkynyl. Particularly suitable are
O((CH.sub.2).sub.nO).sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON((CH.sub.2).sub.nCH.sub.3).sub.2, where n and m
are from 1 to about 10. Other suitable polynucleotides comprise a
sugar substituent group selected from: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl,
O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3,
OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2,
N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving
group, a reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. A suitable
modification includes 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78, 486-504, the disclosure of which is incorporated herein
by reference in its entirety) i.e., an alkoxyalkoxy group. A
further suitable modification includes 2'-dimethylaminooxyethoxy,
i.e., a O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as
2'-DMAOE, as described in examples hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.3).sub.2.
[0308] Other suitable sugar substituent groups include methoxy
(--O--CH.sub.3), aminopropoxy
(--OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), allyl
(--CH.sub.2--CH.dbd.CH.sub.2), --O-allyl
(--O--CH.sub.2--CH.dbd.CH.sub.2) and fluoro (F). 2'-sugar
substituent groups may be in the arabino (up) position or ribo
(down) position. A suitable 2'-arabino modification is 2'-F.
Similar modifications may also be made at other positions on the
oligomeric compound, particularly the 3' position of the sugar on
the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and
the 5' position of 5' terminal nucleotide. Oligomeric compounds may
also have sugar mimetics such as cyclobutyl moieties in place of
the pentofuranosyl sugar.
Base Modifications and Substitutions
[0309] A subject nucleic acid may also include nucleobase (often
referred to in the art simply as "base") modifications or
substitutions. As used herein, "unmodified" or "natural"
nucleobases include the purine bases adenine (A) and guanine (G),
and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
Modified nucleobases include other synthetic and natural
nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl
cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and
other alkyl derivatives of adenine and guanine, 2-propyl and other
alkyl derivatives of adenine and guanine, 2-thiouracil,
2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine,
5-propynyl (--C.dbd.C--CH.sub.3) uracil and cytosine and other
alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and
thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines
and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and
other 5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine. Further modified nucleobases include tricyclic
pyrimidines such as phenoxazine cytidine
(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine
cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps
such as a substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole
cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).
[0310] Heterocyclic base moieties may also include those in which
the purine or pyrimidine base is replaced with other heterocycles,
for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and
2-pyridone. Further nucleobases include those disclosed in U.S.
Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of
Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I.,
ed. John Wiley & Sons, 1990, those disclosed by Englisch et
al., Angewandte Chemie, International Edition, 1991, 30, 613, and
those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research
and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed.,
CRC Press, 1993; the disclosures of which are incorporated herein
by reference in their entirety. Certain of these nucleobases are
useful for increasing the binding affinity of an oligomeric
compound. These include 5-substituted pyrimidines, 6-azapyrimidines
and N-2, N-6 and 0-6 substituted purines, including
2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
5-methylcytosine substitutions have been shown to increase nucleic
acid duplex stability by 0.6-1.2.degree. C. (Sanghvi et al., eds.,
Antisense Research and Applications, CRC Press, Boca Raton, 1993,
pp. 276-278; the disclosure of which is incorporated herein by
reference in its entirety) and are suitable base substitutions,
e.g., when combined with 2'-O-methoxyethyl sugar modifications.
Conjugates
[0311] Another possible modification of a subject nucleic acid
involves chemically linking to the polynucleotide one or more
moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. These
moieties or conjugates can include conjugate groups covalently
bound to functional groups such as primary or secondary hydroxyl
groups. Conjugate groups include, but are not limited to,
intercalators, reporter molecules, polyamines, polyamides,
polyethylene glycols, polyethers, groups that enhance the
pharmacodynamic properties of oligomers, and groups that enhance
the pharmacokinetic properties of oligomers. Suitable conjugate
groups include, but are not limited to, cholesterols, lipids,
phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and
dyes. Groups that enhance the pharmacodynamic properties include
groups that improve uptake, enhance resistance to degradation,
and/or strengthen sequence-specific hybridization with the target
nucleic acid. Groups that enhance the pharmacokinetic properties
include groups that improve uptake, distribution, metabolism or
excretion of a subject nucleic acid.
[0312] Conjugate moieties include but are not limited to lipid
moieties such as a cholesterol moiety (Letsinger et al., Proc.
Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan
et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether,
e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci.,
1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let.,
1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl.
Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,
dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,
1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,
327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a
phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937).
[0313] A conjugate may include a "Protein Transduction Domain" or
PTD (also known as a CPP--cell penetrating peptide), which may
refer to a polypeptide, polynucleotide, carbohydrate, or organic or
inorganic compound that facilitates traversing a lipid bilayer,
micelle, cell membrane, organelle membrane, or vesicle membrane. A
PTD attached to another molecule, which can range from a small
polar molecule to a large macromolecule and/or a nanoparticle,
facilitates the molecule traversing a membrane, for example going
from extracellular space to intracellular space, or cytosol to
within an organelle (e.g., the nucleus). In some embodiments, a PTD
is covalently linked to the 3' end of an exogenous polynucleotide.
In some embodiments, a PTD is covalently linked to the 5' end of an
exogenous polynucleotide. Exemplary PTDs include but are not
limited to a minimal undecapeptide protein transduction domain
(corresponding to residues 47-57 of HIV-1 TAT comprising
YGRKKRRQRRR; SEQ ID NO: 64); a polyarginine sequence comprising a
number of arginines sufficient to direct entry into a cell (e.g.,
3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender
et al. (2002) Cancer Gene Ther. 9(6):489-96); an Drosophila
Antennapedia protein transduction domain (Noguchi et al. (2003)
Diabetes 52(7):1732-1737); a truncated human calcitonin peptide
(Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine
(Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008);
RRQRRTSKLMKR SEQ ID NO: 65); Transportan
GWTLNSAGYLLGKINLKALAALAKKIL SEQ ID NO: 66);
KALAWEAKLAKALAKALAKHLAKALAKALKCEA SEQ ID NO: 67); and
RQIKIWFQNRRMKWKK SEQ ID NO: 68). Exemplary PTDs include but are not
limited to, YGRKKRRQRRR SEQ ID NO: 64), RKKRRQRRR SEQ ID NO: 69);
an arginine homopolymer of from 3 arginine residues to 50 arginine
residues; Exemplary PTD domain amino acid sequences include, but
are not limited to, any of the following: YGRKKRRQRRR SEQ ID NO:
64); RKKRRQRR SEQ ID NO: 69); YARAAARQARA SEQ ID NO: 71);
THRLPRRRRRR SEQ ID NO: 72); and GGRRARRRRRR SEQ ID NO: 73). In some
embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al.
(2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPPs comprise a
polycationic CPP (e.g., Arg9 or "R9") connected via a cleavable
linker to a matching polyanion (e.g., Glu9 or "E9"), which reduces
the net charge to nearly zero and thereby inhibits adhesion and
uptake into cells. Upon cleavage of the linker, the polyanion is
released, locally unmasking the polyarginine and its inherent
adhesiveness, thus "activating" the ACPP to traverse the
membrane.
Introducing Components into a Target Cell
[0314] A Cas12J guide RNA (or a nucleic acid comprising a
nucleotide sequence encoding same) and/or a Cas12J polypeptide of
the present disclosure (or a nucleic acid comprising a nucleotide
sequence encoding same) and/or a Cas12J fusion polypeptide of the
present disclosure (or a nucleic acid that includes a nucleotide
sequence encoding a Cas12J fusion polypeptide of the present
disclosure) and/or a donor polynucleotide (donor template) can be
introduced into a host cell by any of a variety of well-known
methods.
[0315] Any of a variety of compounds and methods can be used to
deliver to a target cell a Cas12J system of the present disclosure
(e.g., where a Cas12J system comprises: a) a Cas12J polypeptide of
the present disclosure and a Cas12J guide RNA; b) a Cas12J
polypeptide of the present disclosure, a Cas12J guide RNA, and a
donor template nucleic acid; c) a Cas12J fusion polypeptide of the
present disclosure and a Cas12J guide RNA; d) a Cas12J fusion
polypeptide of the present disclosure, a Cas12J guide RNA, and a
donor template nucleic acid; e) an mRNA encoding a Cas12J
polypeptide of the present disclosure; and a Cas12J guide RNA; f)
an mRNA encoding a Cas12J polypeptide of the present disclosure, a
Cas12J guide RNA, and a donor template nucleic acid; g) an mRNA
encoding a Cas12J fusion polypeptide of the present disclosure; and
a Cas12J guide RNA; h) an mRNA encoding a Cas12J fusion polypeptide
of the present disclosure, a Cas12J guide RNA, and a donor template
nucleic acid; i) a recombinant expression vector comprising a
nucleotide sequence encoding a Cas12J polypeptide of the present
disclosure and a nucleotide sequence encoding a Cas12J guide RNA;
j) a recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J polypeptide of the present disclosure, a
nucleotide sequence encoding a Cas12J guide RNA, and a nucleotide
sequence encoding a donor template nucleic acid; k) a recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J fusion polypeptide of the present disclosure and a
nucleotide sequence encoding a Cas12J guide RNA; 1) a recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J fusion polypeptide of the present disclosure, a nucleotide
sequence encoding a Cas12J guide RNA, and a nucleotide sequence
encoding a donor template nucleic acid; m) a first recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J polypeptide of the present disclosure, and a second
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J guide RNA; n) a first recombinant expression
vector comprising a nucleotide sequence encoding a Cas12J
polypeptide of the present disclosure, and a second recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J guide RNA; and a donor template nucleic acid; o) a first
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J fusion polypeptide of the present disclosure, and
a second recombinant expression vector comprising a nucleotide
sequence encoding a Cas12J guide RNA; p) a first recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J fusion polypeptide of the present disclosure, and a second
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J guide RNA; and a donor template nucleic acid; q)
a recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J polypeptide of the present disclosure, a
nucleotide sequence encoding a first Cas12J guide RNA, and a
nucleotide sequence encoding a second Cas12J guide RNA; or r) a
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J fusion polypeptide of the present disclosure, a
nucleotide sequence encoding a first Cas12J guide RNA, and a
nucleotide sequence encoding a second Cas12J guide RNA; or some
variation of one of (a) through (r). As a non-limiting example, a
Cas12J system of the present disclosure can be combined with a
lipid. As another non-limiting example, a Cas12J system of the
present disclosure can be combined with a particle, or formulated
into a particle.
[0316] Methods of introducing a nucleic acid into a host cell are
known in the art, and any convenient method can be used to
introduce a subject nucleic acid (e.g., an expression
construct/vector) into a target cell (e.g., prokaryotic cell,
eukaryotic cell, plant cell, animal cell, mammalian cell, human
cell, and the like). Suitable methods include, e.g., viral
infection, transfection, conjugation, protoplast fusion,
lipofection, electroporation, calcium phosphate precipitation,
polyethyleneimine (PEI)-mediated transfection, DEAE-dextran
mediated transfection, liposome-mediated transfection, particle gun
technology, calcium phosphate precipitation, direct micro
injection, nanoparticle-mediated nucleic acid delivery (see, e.g.,
Panyam et., al Adv Drug Deliv Rev. 2012 Sep. 13. pii:
50169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023), and the
like.
[0317] In some cases, a Cas12J polypeptide of the present
disclosure is provided as a nucleic acid (e.g., an mRNA, a DNA, a
plasmid, an expression vector, a viral vector, etc.) that encodes
the Cas12J polypeptide. In some cases, the Cas12J polypeptide of
the present disclosure is provided directly as a protein (e.g.,
without an associated guide RNA or with an associate guide RNA,
i.e., as a ribonucleoprotein complex). A Cas12J polypeptide of the
present disclosure can be introduced into a cell (provided to the
cell) by any convenient method; such methods are known to those of
ordinary skill in the art. As an illustrative example, a Cas12J
polypeptide of the present disclosure can be injected directly into
a cell (e.g., with or without a Cas12J guide RNA or nucleic acid
encoding a Cas12J guide RNA, and with or without a donor
polynucleotide). As another example, a preformed complex of a
Cas12J polypeptide of the present disclosure and a Cas12J guide RNA
(an RNP) can be introduced into a cell (e.g., eukaryotic cell)
(e.g., via injection, via nucleofection; via a protein transduction
domain (PTD) conjugated to one or more components, e.g., conjugated
to the Cas12J protein, conjugated to a guide RNA, conjugated to a
Cas12J polypeptide of the present disclosure and a guide RNA;
etc.).
[0318] In some cases, a Cas12J fusion polypeptide (e.g., dCas12J
fused to a fusion partner, nickase Cas12J fused to a fusion
partner, etc.) of the present disclosure is provided as a nucleic
acid (e.g., an mRNA, a DNA, a plasmid, an expression vector, a
viral vector, etc.) that encodes the Cas12J fusion polypeptide. In
some cases, the Cas12J fusion polypeptide of the present disclosure
is provided directly as a protein (e.g., without an associated
guide RNA or with an associate guide RNA, i.e., as a
ribonucleoprotein complex). A Cas12J fusion polypeptide of the
present disclosure can be introduced into a cell (provided to the
cell) by any convenient method; such methods are known to those of
ordinary skill in the art. As an illustrative example, a Cas12J
fusion polypeptide of the present disclosure can be injected
directly into a cell (e.g., with or without nucleic acid encoding a
Cas12J guide RNA and with or without a donor polynucleotide). As
another example, a preformed complex of a Cas12J fusion polypeptide
of the present disclosure and a Cas12J guide RNA (an RNP) can be
introduced into a cell (e.g., via injection, via nucleofection; via
a protein transduction domain (PTD) conjugated to one or more
components, e.g., conjugated to the Cas12J fusion protein,
conjugated to a guide RNA, conjugated to a Cas12J fusion
polypeptide of the present disclosure and a guide RNA; etc.).
[0319] In some cases, a nucleic acid (e.g., a Cas12J guide RNA; a
nucleic acid comprising a nucleotide sequence encoding a Cas12J
polypeptide of the present disclosure; etc.) is delivered to a cell
(e.g., a target host cell) and/or a polypeptide (e.g., a Cas12J
polypeptide; a Cas12J fusion polypeptide) in a particle, or
associated with a particle. In some cases, a Cas12J system of the
present disclosure is delivered to a cell in a particle, or
associated with a particle. The terms "particle" and nanoparticle"
can be used interchangeable, as appropriate. A recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J polypeptide of the present disclosure and/or a Cas12J guide
RNA, an mRNA comprising a nucleotide sequence encoding a Cas12J
polypeptide of the present disclosure, and guide RNA may be
delivered simultaneously using particles or lipid envelopes; for
instance, a Cas12J polypeptide and a Cas12J guide RNA, e.g., as a
complex (e.g., a ribonucleoprotein (RNP) complex), can be delivered
via a particle, e.g., a delivery particle comprising lipid or
lipidoid and hydrophilic polymer, e.g., a cationic lipid and a
hydrophilic polymer, for instance wherein the cationic lipid
comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or
1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or
wherein the hydrophilic polymer comprises ethylene glycol or
polyethylene glycol (PEG); and/or wherein the particle further
comprises cholesterol (e.g., particle from formulation 1=DOTAP 100,
DMPC 0, PEG 0, Cholesterol 0; formulation number 2=DOTAP 90, DMPC
0, PEG 10, Cholesterol 0; formulation number 3=DOTAP 90, DMPC 0,
PEG 5, Cholesterol 5). For example, a particle can be formed using
a multistep process in which a Cas12J polypepide and a Cas12J
guideRNA are mixed together, e.g., at a 1:1 molar ratio, e.g., at
room temperature, e.g., for 30 minutes, e.g., in sterile, nuclease
free 1.times.phosphate-buffered saline (PBS); and separately,
DOTAP, DMPC, PEG, and cholesterol as applicable for the formulation
are dissolved in alcohol, e.g., 100% ethanol; and, the two
solutions are mixed together to form particles containing the
complexes).
[0320] A Cas12J polypeptide of the present disclosure (or an mRNA
comprising a nucleotide sequence encoding a Cas12J polypeptide of
the present disclosure; or a recombinant expression vector
comprising a nucleotide sequence encoding a Cas12J polypeptide of
the present disclosure) and/or Cas12J guide RNA (or a nucleic acid
such as one or more expression vectors encoding the Cas12J guide
RNA) may be delivered simultaneously using particles or lipid
envelopes. For example, a biodegradable core-shell structured
nanoparticle with a poly (.beta.-amino ester) (PBAE) core enveloped
by a phospholipid bilayer shell can be used. In some cases,
particles/nanoparticles based on self assembling bioadhesive
polymers are used; such particles/nanoparticles may be applied to
oral delivery of peptides, intravenous delivery of peptides and
nasal delivery of peptides, e.g., to the brain. Other embodiments,
such as oral absorption and ocular delivery of hydrophobic drugs
are also contemplated. A molecular envelope technology, which
involves an engineered polymer envelope which is protected and
delivered to the site of the disease, can be used. Doses of about 5
mg/kg can be used, with single or multiple doses, depending on
various factors, e.g., the target tissue.
[0321] Lipidoid compounds (e.g., as described in US patent
application 20110293703) are also useful in the administration of
polynucleotides, and can be used to deliver a Cas12J polypeptide of
the present disclosure, a Cas12J fusion polypeptide of the present
disclosure, an RNP of the present disclosure, a nucleic acid of the
present disclosure, or a Cas12J system of the present disclosure
(e.g., where a Cas12J system comprises: a) a Cas12J polypeptide of
the present disclosure and a Cas12J guide RNA; b) a Cas12J
polypeptide of the present disclosure, a Cas12J guide RNA, and a
donor template nucleic acid; c) a Cas12J fusion polypeptide of the
present disclosure and a Cas12J guide RNA; d) a Cas12J fusion
polypeptide of the present disclosure, a Cas12J guide RNA, and a
donor template nucleic acid; e) an mRNA encoding a Cas12J
polypeptide of the present disclosure; and a Cas12J guide RNA; f)
an mRNA encoding a Cas12J polypeptide of the present disclosure, a
Cas12J guide RNA, and a donor template nucleic acid; g) an mRNA
encoding a Cas12J fusion polypeptide of the present disclosure; and
a Cas12J guide RNA; h) an mRNA encoding a Cas12J fusion polypeptide
of the present disclosure, a Cas12J guide RNA, and a donor template
nucleic acid; i) a recombinant expression vector comprising a
nucleotide sequence encoding a Cas12J polypeptide of the present
disclosure and a nucleotide sequence encoding a Cas12J guide RNA;
j) a recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J polypeptide of the present disclosure, a
nucleotide sequence encoding a Cas12J guide RNA, and a nucleotide
sequence encoding a donor template nucleic acid; k) a recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J fusion polypeptide of the present disclosure and a
nucleotide sequence encoding a Cas12J guide RNA; 1) a recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J fusion polypeptide of the present disclosure, a nucleotide
sequence encoding a Cas12J guide RNA, and a nucleotide sequence
encoding a donor template nucleic acid; m) a first recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J polypeptide of the present disclosure, and a second
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J guide RNA; n) a first recombinant expression
vector comprising a nucleotide sequence encoding a Cas12J
polypeptide of the present disclosure, and a second recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J guide RNA; and a donor template nucleic acid; o) a first
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J fusion polypeptide of the present disclosure, and
a second recombinant expression vector comprising a nucleotide
sequence encoding a Cas12J guide RNA; p) a first recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J fusion polypeptide of the present disclosure, and a second
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J guide RNA; and a donor template nucleic acid; q)
a recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J polypeptide of the present disclosure, a
nucleotide sequence encoding a first Cas12J guide RNA, and a
nucleotide sequence encoding a second Cas12J guide RNA; or r) a
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J fusion polypeptide of the present disclosure, a
nucleotide sequence encoding a first Cas12J guide RNA, and a
nucleotide sequence encoding a second Cas12J guide RNA; or some
variation of one of (a) through (r). In one aspect, the
aminoalcohol lipidoid compounds are combined with an agent to be
delivered to a cell or a subject to form microparticles,
nanoparticles, liposomes, or micelles. The aminoalcohol lipidoid
compounds may be combined with other aminoalcohol lipidoid
compounds, polymers (synthetic or natural), surfactants,
cholesterol, carbohydrates, proteins, lipids, etc. to form the
particles. These particles may then optionally be combined with a
pharmaceutical excipient to form a pharmaceutical composition.
[0322] A poly(beta-amino alcohol) (PBAA) can be used to deliver a
Cas12J polypeptide of the present disclosure, a Cas12J fusion
polypeptide of the present disclosure, an RNP of the present
disclosure, a nucleic acid of the present disclosure, or a Cas12J
system of the present disclosure, to a target cell. US Patent
Publication No. 20130302401 relates to a class of poly(beta-amino
alcohols) (PBAAs) that has been prepared using combinatorial
polymerization.
[0323] Sugar-based particles may be used, for example GalNAc, as
described with reference to WO2014118272 (incorporated herein by
reference) and Nair, J K et al., 2014, Journal of the American
Chemical Society 136 (49), 16958-16961) can be used to deliver a
Cas12J polypeptide of the present disclosure, a Cas12J fusion
polypeptide of the present disclosure, an RNP of the present
disclosure, a nucleic acid of the present disclosure, or a Cas12J
system of the present disclosure, to a target cell.
[0324] In some cases, lipid nanoparticles (LNPs) are used to
deliver a Cas12J polypeptide of the present disclosure, a Cas12J
fusion polypeptide of the present disclosure, an RNP of the present
disclosure, a nucleic acid of the present disclosure, or a Cas12J
system of the present disclosure, to a target cell. Negatively
charged polymers such as RNA may be loaded into LNPs at low pH
values (e.g., pH 4) where the ionizable lipids display a positive
charge. However, at physiological pH values, the LNPs exhibit a low
surface charge compatible with longer circulation times. Four
species of ionizable cationic lipids have been focused upon, namely
1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),
1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),
1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and
1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLinKC2-DMA). Preparation of LNPs and is described in, e.g., Rosin
et al. (2011) Molecular Therapy 19:1286-2200). The cationic lipids
1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),
1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),
1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),
1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLinKC2-DMA), (3-o-[2''-(methoxypolyethyleneglycol 2000)
succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), and
R-3-[(.omega.-methoxy-poly(ethylene glycol)2000)
carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be
used. A nucleic acid (e.g., a Cas12J guide RNA; a nucleic acid of
the present disclosure; etc.) may be encapsulated in LNPs
containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic
lipid:DSPC:CHOL: PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar
ratios). In some cases, 0.2% SP-DiOC18 is incorporated.
[0325] Spherical Nucleic Acid (SNA.TM.) constructs and other
nanoparticles (particularly gold nanoparticles) can be used to
deliver a Cas12J polypeptide of the present disclosure, a Cas12J
fusion polypeptide of the present disclosure, an RNP of the present
disclosure, a nucleic acid of the present disclosure, or a Cas12J
system of the present disclosure, to a target cell. See, e.g.,
Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao et al.,
Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970,
Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al.,
Nano Lett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci.
USA. 2012 109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang
et al., J. Am. Chem. Soc. 2012 134:16488-1691, Weintraub, Nature
2013 495:S14-S16, Choi et al., Proc. Natl. Acad. Sci. USA. 2013
110(19): 7625-7630, Jensen et al., Sci. Transl. Med. 5, 209ra152
(2013) and Mirkin, et al., Small, 10:186-192.
[0326] Self-assembling nanoparticles with RNA may be constructed
with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp
(RGD) peptide ligand attached at the distal end of the polyethylene
glycol (PEG).
[0327] In general, a "nanoparticle" refers to any particle having a
diameter of less than 1000 nm. In some cases, nanoparticles
suitable for use in delivering a Cas12J polypeptide of the present
disclosure, a Cas12J fusion polypeptide of the present disclosure,
an RNP of the present disclosure, a nucleic acid of the present
disclosure, or a Cas12J system of the present disclosure, to a
target cell have a diameter of 500 nm or less, e.g., from 25 nm to
35 nm, from 35 nm to 50 nm, from 50 nm to 75 nm, from 75 nm to 100
nm, from 100 nm to 150 nm, from 150 nm to 200 nm, from 200 nm to
300 nm, from 300 nm to 400 nm, or from 400 nm to 500 nm. In some
cases, nanoparticles suitable for use in delivering a Cas12J
polypeptide of the present disclosure, a Cas12J fusion polypeptide
of the present disclosure, an RNP of the present disclosure, a
nucleic acid of the present disclosure, or a Cas12J system of the
present disclosure, to a target cell have a diameter of from 25 nm
to 200 nm. In some cases, nanoparticles suitable for use in
delivering a Cas12J polypeptide of the present disclosure, a Cas12J
fusion polypeptide of the present disclosure, an RNP of the present
disclosure, a nucleic acid of the present disclosure, or a Cas12J
system of the present disclosure, to a target cell have a diameter
of 100 nm or less In some cases, nanoparticles suitable for use in
delivering a Cas12J polypeptide of the present disclosure, a Cas12J
fusion polypeptide of the present disclosure, an RNP of the present
disclosure, a nucleic acid of the present disclosure, or a Cas12J
system of the present disclosure, to a target cell have a diameter
of from 35 nm to 60 nm.
[0328] Nanoparticles suitable for use in delivering a Cas12J
polypeptide of the present disclosure, a Cas12J fusion polypeptide
of the present disclosure, an RNP of the present disclosure, a
nucleic acid of the present disclosure, or a Cas12J system of the
present disclosure, to a target cell may be provided in different
forms, e.g., as solid nanoparticles (e.g., metal such as silver,
gold, iron, titanium), non-metal, lipid-based solids, polymers),
suspensions of nanoparticles, or combinations thereof. Metal,
dielectric, and semiconductor nanoparticles may be prepared, as
well as hybrid structures (e.g., core-shell nanoparticles).
Nanoparticles made of semiconducting material may also be labeled
quantum dots if they are small enough (typically below 10 nm) that
quantization of electronic energy levels occurs. Such nanoscale
particles are used in biomedical applications as drug carriers or
imaging agents and may be adapted for similar purposes in the
present disclosure.
[0329] Semi-solid and soft nanoparticles are also suitable for use
in delivering a Cas12J polypeptide of the present disclosure, a
Cas12J fusion polypeptide of the present disclosure, an RNP of the
present disclosure, a nucleic acid of the present disclosure, or a
Cas12J system of the present disclosure, to a target cell. A
prototype nanoparticle of semi-solid nature is the liposome.
[0330] In some cases, an exosome is used to deliver a Cas12J
polypeptide of the present disclosure, a Cas12J fusion polypeptide
of the present disclosure, an RNP of the present disclosure, a
nucleic acid of the present disclosure, or a Cas12J system of the
present disclosure, to a target cell. Exosomes are endogenous
nano-vesicles that transport RNAs and proteins, and which can
deliver RNA to the brain and other target organs.
[0331] In some cases, a liposome is used to deliver a Cas12J
polypeptide of the present disclosure, a Cas12J fusion polypeptide
of the present disclosure, an RNP of the present disclosure, a
nucleic acid of the present disclosure, or a Cas12J system of the
present disclosure, to a target cell. Liposomes are spherical
vesicle structures composed of a uni- or multilamellar lipid
bilayer surrounding internal aqueous compartments and a relatively
impermeable outer lipophilic phospholipid bilayer. Liposomes can be
made from several different types of lipids; however, phospholipids
are most commonly used to generate liposomes. Although liposome
formation is spontaneous when a lipid film is mixed with an aqueous
solution, it can also be expedited by applying force in the form of
shaking by using a homogenizer, sonicator, or an extrusion
apparatus. Several other additives may be added to liposomes in
order to modify their structure and properties. For instance,
either cholesterol or sphingomyelin may be added to the liposomal
mixture in order to help stabilize the liposomal structure and to
prevent the leakage of the liposomal inner cargo. A liposome
formulation may be mainly comprised of natural phospholipids and
lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline
(DSPC), sphingomyelin, egg phosphatidylcholines and
monosialoganglioside.
[0332] A stable nucleic-acid-lipid particle (SNALP) can be used to
deliver a Cas12J polypeptide of the present disclosure, a Cas12J
fusion polypeptide of the present disclosure, an RNP of the present
disclosure, a nucleic acid of the present disclosure, or a Cas12J
system of the present disclosure, to a target cell. The SNALP
formulation may contain the lipids 3-N-[(methoxypoly(ethylene
glycol) 2000) carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),
1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol,
in a 2:40:10:48 molar percent ratio. The SNALP liposomes may be
prepared by formulating D-Lin-DMA and PEG-C-DMA with
distearoylphosphatidylcholine (DSPC), Cholesterol and siRNA using a
25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio of
Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulting SNALP liposomes
can be about 80-100 nm in size. A SNALP may comprise synthetic
cholesterol (Sigma-Aldrich, St Louis, Mo., USA),
dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster,
Ala., USA), 3-N-[(w-methoxy poly(ethylene
glycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, and cationic
1,2-dilinoleyloxy-3-N,Ndimethylaminopropane. A SNALP may comprise
synthetic cholesterol (Sigma-Aldrich),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar
Lipids Inc.), PEG-cDMA, and
1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA).
[0333] Other cationic lipids, such as amino lipid
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA)
can be used to deliver a Cas12J polypeptide of the present
disclosure, a Cas12J fusion polypeptide of the present disclosure,
an RNP of the present disclosure, a nucleic acid of the present
disclosure, or a Cas12J system of the present disclosure, to a
target cell. A preformed vesicle with the following lipid
composition may be contemplated: amino lipid,
distearoylphosphatidylcholine (DSPC), cholesterol and
(R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethylene
glycol)2000)propylcarbamate (PEG-lipid) in the molar ratio
40/10/40/10, respectively, and a FVII siRNA/total lipid ratio of
approximately 0.05 (w/w). To ensure a narrow particle size
distribution in the range of 70-90 nm and a low polydispersity
index of 0.11.+-0.0.04 (n=56), the particles may be extruded up to
three times through 80 nm membranes prior to adding the guide RNA.
Particles containing the highly potent amino lipid 16 may be used,
in which the molar ratio of the four lipid components 16, DSPC,
cholesterol and PEG-lipid (50/10/38.5/1.5) which may be further
optimized to enhance in vivo activity.
[0334] Lipids may be formulated with a Cas12J system of the present
disclosure or component(s) thereof or nucleic acids encoding the
same to form lipid nanoparticles (LNPs). Suitable lipids include,
but are not limited to, DLin-KC2-DMA4, C12-200 and colipids
disteroylphosphatidyl choline, cholesterol, and PEG-DMG may be
formulated with a Cas12J system, or component thereof, of the
present disclosure, using a spontaneous vesicle formation
procedure. The component molar ratio may be about 50/10/38.5/1.5
(DLin-KC2-DMA or C12-200/disteroylphosphatidyl
choline/cholesterol/PEG-DMG).
[0335] A Cas12J system of the present disclosure, or a component
thereof, may be delivered encapsulated in PLGA microspheres such as
that further described in US published applications 20130252281 and
20130245107 and 20130244279.
[0336] Supercharged proteins can be used to deliver a Cas12J
polypeptide of the present disclosure, a Cas12J fusion polypeptide
of the present disclosure, an RNP of the present disclosure, a
nucleic acid of the present disclosure, or a Cas12J system of the
present disclosure, to a target cell. Supercharged proteins are a
class of engineered or naturally occurring proteins with unusually
high positive or negative net theoretical charge. Both
supernegatively and superpositively charged proteins exhibit the
ability to withstand thermally or chemically induced aggregation.
Superpositively charged proteins are also able to penetrate
mammalian cells. Associating cargo with these proteins, such as
plasmid DNA, RNA, or other proteins, can enable the functional
delivery of these macromolecules into mammalian cells both in vitro
and in vivo.
[0337] Cell Penetrating Peptides (CPPs) can be used to deliver a
Cas12J polypeptide of the present disclosure, a Cas12J fusion
polypeptide of the present disclosure, an RNP of the present
disclosure, a nucleic acid of the present disclosure, or a Cas12J
system of the present disclosure, to a target cell. CPPs typically
have an amino acid composition that either contains a high relative
abundance of positively charged amino acids such as lysine or
arginine or has sequences that contain an alternating pattern of
polar/charged amino acids and non-polar, hydrophobic amino
acids.
[0338] An implantable device can be used to deliver a Cas12J
polypeptide of the present disclosure, a Cas12J fusion polypeptide
of the present disclosure, an RNP of the present disclosure, a
nucleic acid of the present disclosure (e.g., a Cas12J guide RNA, a
nucleic acid encoding a Cas12J guide RNA, a nucleic acid encoding
Cas12J polypeptide, a donor template, and the like), or a Cas12J
system of the present disclosure, to a target cell (e.g., a target
cell in vivo, where the target cell is a target cell in
circulation, a target cell in a tissue, a target cell in an organ,
etc.). An implantable device suitable for use in delivering a
Cas12J polypeptide of the present disclosure, a Cas12J fusion
polypeptide of the present disclosure, an RNP of the present
disclosure, a nucleic acid of the present disclosure, or a Cas12J
system of the present disclosure, to a target cell (e.g., a target
cell in vivo, where the target cell is a target cell in
circulation, a target cell in a tissue, a target cell in an organ,
etc.) can include a container (e.g., a reservoir, a matrix, etc.)
that comprises the Cas12J polypeptide, the Cas12J fusion
polypeptide, the RNP, or the Cas12J system (or component thereof,
e.g., a nucleic acid of the present disclosure).
[0339] A suitable implantable device can comprise a polymeric
substrate, such as a matrix for example, that is used as the device
body, and in some cases additional scaffolding materials, such as
metals or additional polymers, and materials to enhance visibility
and imaging. An implantable delivery device can be advantageous in
providing release locally and over a prolonged period, where the
polypeptide and/or nucleic acid to be delivered is released
directly to a target site, e.g., the extracellular matrix (ECM),
the vasculature surrounding a tumor, a diseased tissue, etc.
Suitable implantable delivery devices include devices suitable for
use in delivering to a cavity such as the abdominal cavity and/or
any other type of administration in which the drug delivery system
is not anchored or attached, comprising a biostable and/or
degradable and/or bioabsorbable polymeric substrate, which may for
example optionally be a matrix. In some cases, a suitable
implantable drug delivery device comprises degradable polymers,
wherein the main release mechanism is bulk erosion. In some cases,
a suitable implantable drug delivery device comprises non
degradable, or slowly degraded polymers, wherein the main release
mechanism is diffusion rather than bulk erosion, so that the outer
part functions as membrane, and its internal part functions as a
drug reservoir, which practically is not affected by the
surroundings for an extended period (for example from about a week
to about a few months). Combinations of different polymers with
different release mechanisms may also optionally be used. The
concentration gradient at the can be maintained effectively
constant during a significant period of the total releasing period,
and therefore the diffusion rate is effectively constant (termed
"zero mode" diffusion). By the term "constant" it is meant a
diffusion rate that is maintained above the lower threshold of
therapeutic effectiveness, but which may still optionally feature
an initial burst and/or may fluctuate, for example increasing and
decreasing to a certain degree. The diffusion rate can be so
maintained for a prolonged period, and it can be considered
constant to a certain level to optimize the therapeutically
effective period, for example the effective silencing period.
[0340] In some cases, the implantable delivery system is designed
to shield the nucleotide based therapeutic agent from degradation,
whether chemical in nature or due to attack from enzymes and other
factors in the body of the subject.
[0341] The site for implantation of the device, or target site, can
be selected for maximum therapeutic efficacy. For example, a
delivery device can be implanted within or in the proximity of a
tumor environment, or the blood supply associated with a tumor. The
target location can be, e.g.: 1) the brain at degenerative sites
like in Parkinson or Alzheimer disease at the basal ganglia, white
and gray matter; 2) the spine, as in the case of amyotrophic
lateral sclerosis (ALS); 3) uterine cervix; 4) active and chronic
inflammatory joints; 5) dermis as in the case of psoriasis; 7)
sympathetic and sensoric nervous sites for analgesic effect; 7) a
bone; 8) a site of acute or chronic infection; 9) Intra vaginal;
10) Inner ear--auditory system, labyrinth of the inner ear,
vestibular system; 11) Intra tracheal; 12) Intra-cardiac; coronary,
epicardiac; 13) urinary tract or bladder; 14) biliary system; 15)
parenchymal tissue including and not limited to the kidney, liver,
spleen; 16) lymph nodes; 17) salivary glands; 18) dental gums; 19)
Intra-articular (into joints); 20) Intra-ocular; 21) Brain tissue;
22) Brain ventricles; 23) Cavities, including abdominal cavity (for
example but without limitation, for ovary cancer); 24) Intra
esophageal; and 25) Intra rectal; and 26) into the vasculature.
[0342] The method of insertion, such as implantation, may
optionally already be used for other types of tissue implantation
and/or for insertions and/or for sampling tissues, optionally
without modifications, or alternatively optionally only with
non-major modifications in such methods. Such methods optionally
include but are not limited to brachytherapy methods, biopsy,
endoscopy with and/or without ultrasound, such as stereotactic
methods into the brain tissue, laparoscopy, including implantation
with a laparoscope into joints, abdominal organs, the bladder wall
and body cavities.
Modified Host Cells
[0343] The present disclosure provides a modified cell comprising a
Cas12J polypeptide of the present disclosure and/or a nucleic acid
comprising a nucleotide sequence encoding a Cas12J polypeptide of
the present disclosure. The present disclosure provides a modified
cell comprising a Cas12J polypeptide of the present disclosure,
where the modified cell is a cell that does not normally comprise a
Cas12J polypeptide of the present disclosure. The present
disclosure provides a modified cell (e.g., a genetically modified
cell) comprising nucleic acid comprising a nucleotide sequence
encoding a Cas12J polypeptide of the present disclosure. The
present disclosure provides a genetically modified cell that is
genetically modified with an mRNA comprising a nucleotide sequence
encoding a Cas12J polypeptide of the present disclosure. The
present disclosure provides a genetically modified cell that is
genetically modified with a recombinant expression vector
comprising a nucleotide sequence encoding a Cas12J polypeptide of
the present disclosure. The present disclosure provides a
genetically modified cell that is genetically modified with a
recombinant expression vector comprising: a) a nucleotide sequence
encoding a Cas12J polypeptide of the present disclosure; and b) a
nucleotide sequence encoding a Cas12J guide RNA of the present
disclosure. The present disclosure provides a genetically modified
cell that is genetically modified with a recombinant expression
vector comprising: a) a nucleotide sequence encoding a Cas12J
polypeptide of the present disclosure; b) a nucleotide sequence
encoding a Cas12J guide RNA of the present disclosure; and c) a
nucleotide sequence encoding a donor template.
[0344] A cell that serves as a recipient for a Cas12J polypeptide
of the present disclosure and/or a nucleic acid comprising a
nucleotide sequence encoding a Cas12J polypeptide of the present
disclosure and/or a Cas12J guide RNA of the present disclosure, can
be any of a variety of cells, including, e.g., in vitro cells; in
vivo cells; ex vivo cells; primary cells; cancer cells; animal
cells; plant cells; algal cells; fungal cells; etc. A cell that
serves as a recipient for a Cas12J polypeptide of the present
disclosure and/or a nucleic acid comprising a nucleotide sequence
encoding a Cas12J polypeptide of the present disclosure and/or a
Cas12J guide RNA of the present disclosure is referred to as a
"host cell" or a "target cell." A host cell or a target cell can be
a recipient of a Cas12J system of the present disclosure. A host
cell or a target cell can be a recipient of a Cas12J RNP of the
present disclosure. A host cell or a target cell can be a recipient
of a single component of a Cas12J system of the present
disclosure.
[0345] Non-limiting examples of cells (target cells) include: a
prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal
cell, a cell of a single-cell eukaryotic organism, a protozoa cell,
a cell from a plant (e.g., cells from plant crops, fruits,
vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes,
rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis,
tobacco, flowering plants, conifers, gymnosperms, angiosperms,
ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons,
monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii,
Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella
pyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds
(e.g. kelp) a fungal cell (e.g., a yeast cell, a cell from a
mushroom), an animal cell, a cell from an invertebrate animal
(e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell
from a vertebrate animal (e.g., fish, amphibian, reptile, bird,
mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, a
cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human
primate; a human; a feline (e.g., a cat); a canine (e.g., a dog);
etc.), and the like. In some cases, the cell is a cell that does
not originate from a natural organism (e.g., the cell can be a
synthetically made cell; also referred to as an artificial
cell).
[0346] A cell can be an in vitro cell (e.g., established cultured
cell line). A cell can be an ex vivo cell (cultured cell from an
individual). A cell can be and in vivo cell (e.g., a cell in an
individual). A cell can be an isolated cell. A cell can be a cell
inside of an organism. A cell can be an organism. A cell can be a
cell in a cell culture (e.g., in vitro cell culture). A cell can be
one of a collection of cells. A cell can be a prokaryotic cell or
derived from a prokaryotic cell. A cell can be a bacterial cell or
can be derived from a bacterial cell. A cell can be an archaeal
cell or derived from an archaeal cell. A cell can be a eukaryotic
cell or derived from a eukaryotic cell. A cell can be a plant cell
or derived from a plant cell. A cell can be an animal cell or
derived from an animal cell. A cell can be an invertebrate cell or
derived from an invertebrate cell. A cell can be a vertebrate cell
or derived from a vertebrate cell. A cell can be a mammalian cell
or derived from a mammalian cell. A cell can be a rodent cell or
derived from a rodent cell. A cell can be a human cell or derived
from a human cell. A cell can be a microbe cell or derived from a
microbe cell. A cell can be a fungi cell or derived from a fungi
cell. A cell can be an insect cell. A cell can be an arthropod
cell. A cell can be a protozoan cell. A cell can be a helminth
cell.
[0347] Suitable cells include a stem cell (e.g. an embryonic stem
(ES) cell, an induced pluripotent stem (iPS) cell; a germ cell
(e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a
somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell,
a hematopoietic cell, a neuron, a muscle cell, a bone cell, a
hepatocyte, a pancreatic cell, etc.
[0348] Suitable cells include human embryonic stem cells, fetal
cardiomyocytes, myofibroblasts, mesenchymal stem cells,
cardiomyocytes, adipocytes, totipotent cells, pluripotent cells,
blood stem cells, myoblasts, adult stem cells, bone marrow cells,
mesenchymal cells, embryonic stem cells, parenchymal cells,
epithelial cells, endothelial cells, mesothelial cells,
fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous
cells, stem cells, hematopoietic stem cells, bone-marrow derived
progenitor cells, myocardial cells, skeletal cells, fetal cells,
undifferentiated cells, multi-potent progenitor cells, unipotent
progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts,
macrophages, capillary endothelial cells, xenogenic cells,
allogenic cells, and post-natal stem cells.
[0349] In some cases, the cell is an immune cell, a neuron, an
epithelial cell, and endothelial cell, or a stem cell. In some
cases, the immune cell is a T cell, a B cell, a monocyte, a natural
killer cell, a dendritic cell, or a macrophage. In some cases, the
immune cell is a cytotoxic T cell. In some cases, the immune cell
is a helper T cell. In some cases, the immune cell is a regulatory
T cell (Treg).
[0350] In some cases, the cell is a stem cell. Stem cells include
adult stem cells. Adult stem cells are also referred to as somatic
stem cells.
[0351] Adult stem cells are resident in differentiated tissue, but
retain the properties of self-renewal and ability to give rise to
multiple cell types, usually cell types typical of the tissue in
which the stem cells are found. Numerous examples of somatic stem
cells are known to those of skill in the art, including muscle stem
cells; hematopoietic stem cells; epithelial stem cells; neural stem
cells; mesenchymal stem cells; mammary stem cells; intestinal stem
cells; mesodermal stem cells; endothelial stem cells; olfactory
stem cells; neural crest stem cells; and the like.
[0352] Stem cells of interest include mammalian stem cells, where
the term "mammalian" refers to any animal classified as a mammal,
including humans; non-human primates; domestic and farm animals;
and zoo, laboratory, sports, or pet animals, such as dogs, horses,
cats, cows, mice, rats, rabbits, etc. In some cases, the stem cell
is a human stem cell. In some cases, the stem cell is a rodent
(e.g., a mouse; a rat) stem cell. In some cases, the stem cell is a
non-human primate stem cell.
[0353] Stem cells can express one or more stem cell markers, e.g.,
SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1,
OLFM4, CDH17, and PPARGC1A.
[0354] In some embodiments, the stem cell is a hematopoietic stem
cell (HSC). HSCs are mesoderm-derived cells that can be isolated
from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs
are characterized as CD34+ and CD3-. HSCs can repopulate the
erythroid, neutrophil-macrophage, megakaryocyte and lymphoid
hematopoietic cell lineages in vivo. In vitro, HSCs can be induced
to undergo at least some self-renewing cell divisions and can be
induced to differentiate to the same lineages as is seen in vivo.
As such, HSCs can be induced to differentiate into one or more of
erythroid cells, megakaryocytes, neutrophils, macrophages, and
lymphoid cells.
[0355] In other embodiments, the stem cell is a neural stem cell
(NSC). Neural stem cells (NSCs) are capable of differentiating into
neurons, and glia (including oligodendrocytes, and astrocytes). A
neural stem cell is a multipotent stem cell which is capable of
multiple divisions, and under specific conditions can produce
daughter cells which are neural stem cells, or neural progenitor
cells that can be neuroblasts or glioblasts, e.g., cells committed
to become one or more types of neurons and glial cells
respectively. Methods of obtaining NSCs are known in the art.
[0356] In other embodiments, the stem cell is a mesenchymal stem
cell (MSC). MSCs originally derived from the embryonal mesoderm and
isolated from adult bone marrow, can differentiate to form muscle,
bone, cartilage, fat, marrow stroma, and tendon. Methods of
isolating MSC are known in the art; and any known method can be
used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which
describes isolation of human MSC.
[0357] A cell is in some cases a plant cell. A plant cell can be a
cell of a monocotyledon. A cell can be a cell of a dicotyledon.
[0358] In some cases, the cell is a plant cell. For example, the
cell can be a cell of a major agricultural plant, e.g., Barley,
Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland),
Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice,
Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil),
Sunflowers (Non-Oil), Sweet Potatoes, Tobacco (Burley), Tobacco
(Flue-cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat
(Winter), and the like. As another example, the cell is a cell of a
vegetable crops which include but are not limited to, e.g., alfalfa
sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus,
bamboo shoots, banana flowers, bean sprouts, beans, beet tops,
beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini),
brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales),
calabaza, cardoon, carrots, cauliflower, celery, chayote, chinese
artichoke (crosnes), chinese cabbage, chinese celery, chinese
chives, choy sum, chrysanthemum leaves (tung ho), collard greens,
corn stalks, corn-sweet, cucumbers, daikon, dandelion greens,
dasheen, dau mue (pea tips), donqua (winter melon), eggplant,
endive, escarole, fiddle head ferns, field cress, frisee, gai choy
(chinese mustard), gailon, galanga (siam, thai ginger), garlic,
ginger root, gobo, greens, hanover salad greens, huauzontle,
jerusalem artichokes, jicama, kale greens, kohlrabi, lamb's
quarters (quilete), lettuce (bibb), lettuce (boston), lettuce
(boston red), lettuce (green leaf), lettuce (iceberg), lettuce
(lolla rossa), lettuce (oak leaf--green), lettuce (oak leaf--red),
lettuce (processed), lettuce (red leaf), lettuce (romaine), lettuce
(ruby romaine), lettuce (russian red mustard), linkok, lo bok, long
beans, lotus root, mache, maguey (agave) leaves, malanga, mesculin
mix, mizuna, moap (smooth luffa), moo, moqua (fuzzy squash),
mushrooms, mustard, nagaimo, okra, ong choy, onions green, opo
(long squash), ornamental corn, ornamental gourds, parsley,
parsnips, peas, peppers (bell type), peppers, pumpkins, radicchio,
radish sprouts, radishes, rape greens, rape greens, rhubarb,
romaine (baby red), rutabagas, salicornia (sea bean), sinqua
(angled/ridged luffa), spinach, squash, straw bales, sugarcane,
sweet potatoes, swiss chard, tamarindo, taro, taro leaf, taro
shoots, tatsoi, tepeguaje (guaje), tindora, tomatillos, tomatoes,
tomatoes (cherry), tomatoes (grape type), tomatoes (plum type),
tumeric, turnip tops greens, turnips, water chestnuts, yampi, yams
(names), yu choy, yuca (cassava), and the like.
[0359] In some cases, the plant cell is a cell of a plant component
such as a leaf, a stem, a root, a seed, a flower, pollen, an
anther, an ovule, a pedicel, a fruit, a meristem, a cotyledon, a
hypocotyl, a pod, an embryo, endosperm, an explant, a callus, or a
shoot.
[0360] A cell is in some cases an arthropod cell. For example, the
cell can be a cell of a sub-order, a family, a sub-family, a group,
a sub-group, or a species of, e.g., Chelicerata, Myriapodia,
Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura,
Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera,
Neoptera, Exopterygota, Plecoptera, Embioptera, Orthoptera,
Zoraptera, Dermaptera, Dictyoptera, Notoptera, Grylloblattidae,
Mantophasmatidae, Phasmatodea, Blattaria, Isoptera, Mantodea,
Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera,
Endopterygota or Holometabola, Hymenoptera, Coleoptera,
Strepsiptera, Raphidioptera, Megaloptera, Neuroptera, Mecoptera,
Siphonaptera, Diptera, Trichoptera, or Lepidoptera.
[0361] A cell is in some cases an insect cell. For example, in some
cases, the cell is a cell of a mosquito, a grasshopper, a true bug,
a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a
beetle.
Kits
[0362] The present disclosure provides a kit comprising a Cas12J
system of the present disclosure, or a component of a Cas12J system
of the present disclosure.
[0363] A kit of the present disclosure can comprise: a) a Cas12J
polypeptide of the present disclosure and a Cas12J guide RNA; b) a
Cas12J polypeptide of the present disclosure, a Cas12J guide RNA,
and a donor template nucleic acid; c) a Cas12J fusion polypeptide
of the present disclosure and a Cas12J guide RNA; d) a Cas12J
fusion polypeptide of the present disclosure, a Cas12J guide RNA,
and a donor template nucleic acid; e) an mRNA encoding a Cas12J
polypeptide of the present disclosure; and a Cas12J guide RNA; f)
an mRNA encoding a Cas12J polypeptide of the present disclosure, a
Cas12J guide RNA, and a donor template nucleic acid; g) an mRNA
encoding a Cas12J fusion polypeptide of the present disclosure; and
a Cas12J guide RNA; h) an mRNA encoding a Cas12J fusion polypeptide
of the present disclosure, a Cas12J guide RNA, and a donor template
nucleic acid; i) a recombinant expression vector comprising a
nucleotide sequence encoding a Cas12J polypeptide of the present
disclosure and a nucleotide sequence encoding a Cas12J guide RNA;
j) a recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J polypeptide of the present disclosure, a
nucleotide sequence encoding a Cas12J guide RNA, and a nucleotide
sequence encoding a donor template nucleic acid; k) a recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J fusion polypeptide of the present disclosure and a
nucleotide sequence encoding a Cas12J guide RNA; l) a recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J fusion polypeptide of the present disclosure, a nucleotide
sequence encoding a Cas12J guide RNA, and a nucleotide sequence
encoding a donor template nucleic acid; m) a first recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J polypeptide of the present disclosure, and a second
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J guide RNA; n) a first recombinant expression
vector comprising a nucleotide sequence encoding a Cas12J
polypeptide of the present disclosure, and a second recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J guide RNA; and a donor template nucleic acid; o) a first
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J fusion polypeptide of the present disclosure, and
a second recombinant expression vector comprising a nucleotide
sequence encoding a Cas12J guide RNA; p) a first recombinant
expression vector comprising a nucleotide sequence encoding a
Cas12J fusion polypeptide of the present disclosure, and a second
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J guide RNA; and a donor template nucleic acid; q)
a recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J polypeptide of the present disclosure, a
nucleotide sequence encoding a first Cas12J guide RNA, and a
nucleotide sequence encoding a second Cas12J guide RNA; or r) a
recombinant expression vector comprising a nucleotide sequence
encoding a Cas12J fusion polypeptide of the present disclosure, a
nucleotide sequence encoding a first Cas12J guide RNA, and a
nucleotide sequence encoding a second Cas12J guide RNA; or some
variation of one of (a) through (r).
[0364] A kit of the present disclosure can comprise: a) a
component, as described above, of a Cas12J system of the present
disclosure, or can comprise a Cas12J system of the present
disclosure; and b) one or more additional reagents, e.g., i) a
buffer; ii) a protease inhibitor; iii) a nuclease inhibitor; iv) a
reagent required to develop or visualize a detectable label; v) a
positive and/or negative control target DNA; vi) a positive and/or
negative control Cas12J guide RNA; and the like. A kit of the
present disclosure can comprise: a) a component, as described
above, of a Cas12J system of the present disclosure, or can
comprise a Cas12J system of the present disclosure; and b) a
therapeutic agent.
[0365] A kit of the present disclosure can comprise a recombinant
expression vector comprising: a) an insertion site for inserting a
nucleic acid comprising a nucleotide sequence encoding a portion of
a Cas12J guide RNA that hybridizes to a target nucleotide sequence
in a target nucleic acid; and b) a nucleotide sequence encoding the
Cas12J-binding portion of a Cas12J guide RNA. A kit of the present
disclosure can comprise a recombinant expression vector comprising:
a) an insertion site for inserting a nucleic acid comprising a
nucleotide sequence encoding a portion of a Cas12J guide RNA that
hybridizes to a target nucleotide sequence in a target nucleic
acid; b) a nucleotide sequence encoding the Cas12J-binding portion
of a Cas12J guide RNA; and c) a nucleotide sequence encoding a
Cas12J polypeptide of the present disclosure.
Utility
[0366] A Cas12J polypeptide of the present disclosure, or a Cas12J
fusion polypeptide of the present disclosure, finds use in a
variety of methods (e.g., in combination with a Cas12J guide RNA
and in some cases further in combination with a donor template).
For example, a Cas12J polypeptide of the present disclosure can be
used to (i) modify (e.g., cleave, e.g., nick; methylate; etc.)
target nucleic acid (DNA or RNA; single stranded or double
stranded); (ii) modulate transcription of a target nucleic acid;
(iii) label a target nucleic acid; (iv) bind a target nucleic acid
(e.g., for purposes of isolation, labeling, imaging, tracking,
etc.); (v) modify a polypeptide (e.g., a histone) associated with a
target nucleic acid; and the like. Thus, the present disclosure
provides a method of modifying a target nucleic acid. In some
cases, a method of the present disclosure for modifying a target
nucleic acid comprises contacting the target nucleic acid with: a)
a Cas12J polypeptide of the present disclosure; and b) one or more
(e.g., two) Cas12J guide RNAs. In some cases, a method of the
present disclosure for modifying a target nucleic acid comprises
contacting the target nucleic acid with: a) a Cas12J polypeptide of
the present disclosure; b) a Cas12J guide RNA; and c) a donor
nucleic acid (e.g., a donor template). In some cases, the
contacting step is carried out in a cell in vitro. In some cases,
the contacting step is carried out in a cell in vivo. In some
cases, the contacting step is carried out in a cell ex vivo.
[0367] Because a method that uses a Cas12J polypeptide includes
binding of the Cas12J polypeptide to a particular region in a
target nucleic acid (by virtue of being targeted there by an
associated Cas12J guide RNA), the methods are generally referred to
herein as methods of binding (e.g., a method of binding a target
nucleic acid). However, it is to be understood that in some cases,
while a method of binding may result in nothing more than binding
of the target nucleic acid, in other cases, the method can have
different final results (e.g., the method can result in
modification of the target nucleic acid, e.g.,
cleavage/methylation/etc., modulation of transcription from the
target nucleic acid; modulation of translation of the target
nucleic acid; genome editing; modulation of a protein associated
with the target nucleic acid; isolation of the target nucleic acid;
etc.).
[0368] For examples of suitable methods, see, for example, Jinek et
al., Science. 2012 Aug. 17; 337(6096):816-21; Chylinski et al., RNA
Biol. 2013 May; 10(5):726-37; Ma et al., Biomed Res Int. 2013;
2013:270805; Hou et al., Proc Natl Acad Sci USA. 2013 Sep. 24;
110(39):15644-9; Jinek et al., Elife. 2013; 2:e00471; Pattanayak et
al., Nat Biotechnol. 2013 September; 31(9):839-43; Qi et al, Cell.
2013 Feb. 28; 152(5):1173-83; Wang et al., Cell. 2013 May 9;
153(4):910-8; Auer et al., Genome Res. 2013 Oct. 31; Chen et al.,
Nucleic Acids Res. 2013 Nov. 1; 41(20):e19; Cheng et al., Cell Res.
2013 October; 23(10):1163-71; Cho et al., Genetics. 2013 November;
195(3):1177-80; DiCarlo et al., Nucleic Acids Res. 2013 April;
41(7):4336-43; Dickinson et al., Nat Methods. 2013 October;
10(10):1028-34; Ebina et al., Sci Rep. 2013; 3:2510; Fujii et al,
Nucleic Acids Res. 2013 Nov. 1; 41(20):e187; Hu et al., Cell Res.
2013 November; 23(11):1322-5; Jiang et al., Nucleic Acids Res. 2013
Nov. 1; 41(20):e188; Larson et al., Nat Protoc. 2013 November;
8(11):2180-96; Mali et. at., Nat Methods. 2013 October;
10(10):957-63; Nakayama et al., Genesis. 2013 December;
51(12):835-43; Ran et al., Nat Protoc. 2013 November;
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154(6):1370-9; and U.S. patents and patent applications: U.S. Pat.
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8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797;
20140170753; 20140179006; 20140179770; 20140186843; 20140186919;
20140186958; 20140189896; 20140227787; 20140234972; 20140242664;
20140242699; 20140242700; 20140242702; 20140248702; 20140256046;
20140273037; 20140273226; 20140273230; 20140273231; 20140273232;
20140273233; 20140273234; 20140273235; 20140287938; 20140295556;
20140295557; 20140298547; 20140304853; 20140309487; 20140310828;
20140310830; 20140315985; 20140335063; 20140335620; 20140342456;
20140342457; 20140342458; 20140349400; 20140349405; 20140356867;
20140356956; 20140356958; 20140356959; 20140357523; 20140357530;
20140364333; and 20140377868; each of which is hereby incorporated
by reference in its entirety.
[0369] For example, the present disclosure provides (but is not
limited to) methods of cleaving a target nucleic acid; methods of
editing a target nucleic acid; methods of modulating transcription
from a target nucleic acid; methods of isolating a target nucleic
acid, methods of binding a target nucleic acid, methods of imaging
a target nucleic acid, methods of modifying a target nucleic acid,
and the like.
[0370] As used herein, the terms/phrases "contact a target nucleic
acid" and "contacting a target nucleic acid", for example, with a
Cas12J polypeptide or with a Cas12J fusion polypeptide, etc.,
encompass all methods for contacting the target nucleic acid. For
example, a Cas12J polypeptide can be provided to a cell as protein,
RNA (encoding the Cas12J polypeptide), or DNA (encoding the Cas12J
polypeptide); while a Cas12J guide RNA can be provided as a guide
RNA or as a nucleic acid encoding the guide RNA. As such, when, for
example, performing a method in a cell (e.g., inside of a cell in
vitro, inside of a cell in vivo, inside of a cell ex vivo), a
method that includes contacting the target nucleic acid encompasses
the introduction into the cell of any or all of the components in
their active/final state (e.g., in the form of a protein(s) for
Cas12J polypeptide; in the form of a protein for a Cas12J fusion
polypeptide; in the form of an RNA in some cases for the guide
RNA), and also encompasses the introduction into the cell of one or
more nucleic acids encoding one or more of the components (e.g.,
nucleic acid(s) comprising nucleotide sequence(s) encoding a Cas12J
polypeptide or a Cas12J fusion polypeptide, nucleic acid(s)
comprising nucleotide sequence(s) encoding guide RNA(s), nucleic
acid comprising a nucleotide sequence encoding a donor template,
and the like). Because the methods can also be performed in vitro
outside of a cell, a method that includes contacting a target
nucleic acid, (unless otherwise specified) encompasses contacting
outside of a cell in vitro, inside of a cell in vitro, inside of a
cell in vivo, inside of a cell ex vivo, etc.
[0371] In some cases, a method of the present disclosure for
modifying a target nucleic acid comprises introducing into a target
cell a Cas12J locus, e.g., a nucleic acid comprising a nucleotide
sequence encoding a Cas12J polypeptide as well as nucleotide
sequences of about 1 kilobase (kb) to 5 kb in length surrounding
the Cas12J-encoding nucleotide sequence from a cell (e.g., in some
cases a cell that in its natural state (the state in which it
occurs in nature) comprises a Cas12J locus) comprising a Cas12J
locus, where the target cell does not normally (in its natural
state) comprise a Cas12J locus. However, one or more spacer
sequences, encoding guide sequences for the encoded crRNA(s), can
be modified such that one or more target sequences of interest are
targeted. Thus, for example, in some cases, a method of the present
disclosure for modifying a target nucleic acid comprises
introducing into a target cell a Cas12J locus, e.g., a nucleic acid
obtained from a source cell (e.g., in some cases a cell that in its
natural state (the state in which it occurs in nature) comprises a
Cas12J locus), where the nucleic acid has a length of from 100
nucleotides (nt) to 5 kb in length (e.g., from 100 nt to 500 nt,
from 500 nt to 1 kb, from 1 kb to 1.5 kb, from 1.5 kb to 2 kb, from
2 kb to 2.5 kb, from 2.5 kb to 3 kb, from 3 kb to 3.5 kb, from 3.5
kb to 4 kb, or from 4 kb to 5 kb in length) and comprises a
nucleotide sequence encoding a Cas12J polypeptide. As noted above,
in some such cases, one or more spacer sequences, encoding guide
sequences for the encoded crRNA(s), can be modified such that one
or more target sequences of interest are targeted. In some cases,
the method comprises introducing into a target cell: i) a Cas12J
locus; and ii) a donor DNA template. In some cases, the target
nucleic acid is in a cell-free composition in vitro. In some cases,
the target nucleic acid is present in a target cell. In some cases,
the target nucleic acid is present in a target cell, where the
target cell is a prokaryotic cell. In some cases, the target
nucleic acid is present in a target cell, where the target cell is
a eukaryotic cell. In some cases, the target nucleic acid is
present in a target cell, where the target cell is a mammalian
cell. In some cases, the target nucleic acid is present in a target
cell, where the target cell is a plant cell.
[0372] In some cases, a method of the present disclosure for
modifying a target nucleic acid comprises contacting a target
nucleic acid with a Cas12J polypeptide of the present disclosure,
or with a Cas12J fusion polypeptide of the present disclosure. In
some cases, a method of the present disclosure for modifying a
target nucleic acid comprises contacting a target nucleic acid with
a Cas12J polypeptide and a Cas12J guide RNA. In some cases, a
method of the present disclosure for modifying a target nucleic
acid comprises contacting a target nucleic acid with a Cas12J
polypeptide, a first Cas12J guide RNA, and a second Cas12J guide
RNA In some cases, a method of the present disclosure for modifying
a target nucleic acid comprises contacting a target nucleic acid
with a Cas12J polypeptide of the present disclosure and a Cas12J
guide RNA and a donor DNA template.
Target Nucleic Acids and Target Cells of Interest
[0373] A Cas12J polypeptide of the present disclosure, or a Cas12J
fusion polypeptide of the present disclosure, when bound to a
Cas12J guide RNA, can bind to a target nucleic acid, and in some
cases, can bind to and modify a target nucleic acid. A target
nucleic acid can be any nucleic acid (e.g., DNA, RNA), can be
double stranded or single stranded, can be any type of nucleic acid
(e.g., a chromosome (genomic DNA), derived from a chromosome,
chromosomal DNA, plasmid, viral, extracellular, intracellular,
mitochondrial, chloroplast, linear, circular, etc.) and can be from
any organism (e.g., as long as the Cas12J guide RNA comprises a
nucleotide sequence that hybridizes to a target sequence in a
target nucleic acid, such that the target nucleic acid can be
targeted).
[0374] A target nucleic acid can be DNA or RNA. A target nucleic
acid can be double stranded (e.g., dsDNA, dsRNA) or single stranded
(e.g., ssRNA, ssDNA). In some cases, a target nucleic acid is
single stranded. In some cases, a target nucleic acid is a single
stranded RNA (ssRNA). In some cases, a target ssRNA (e.g., a target
cell ssRNA, a viral ssRNA, etc.) is selected from: mRNA, rRNA,
tRNA, non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and
microRNA (miRNA). In some cases, a target nucleic acid is a single
stranded DNA (ssDNA) (e.g., a viral DNA). As noted above, in some
cases, a target nucleic acid is single stranded.
[0375] A target nucleic acid can be located anywhere, for example,
outside of a cell in vitro, inside of a cell in vitro, inside of a
cell in vivo, inside of a cell ex vivo. Suitable target cells
(which can comprise target nucleic acids such as genomic DNA)
include, but are not limited to: a bacterial cell; an archaeal
cell; a cell of a single-cell eukaryotic organism; a plant cell; an
algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii,
Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens,
C. agardh, and the like; a fungal cell (e.g., a yeast cell); an
animal cell; a cell from an invertebrate animal (e.g. fruit fly, a
cnidarian, an echinoderm, a nematode, etc.); a cell of an insect
(e.g., a mosquito; a bee; an agricultural pest; etc.); a cell of an
arachnid (e.g., a spider; a tick; etc.); a cell from a vertebrate
animal (e.g., a fish, an amphibian, a reptile, a bird, a mammal); a
cell from a mammal (e.g., a cell from a rodent; a cell from a
human; a cell of a non-human mammal; a cell of a rodent (e.g., a
mouse, a rat); a cell of a lagomorph (e.g., a rabbit); a cell of an
ungulate (e.g., a cow, a horse, a camel, a llama, a vicuna, a
sheep, a goat, etc.); a cell of a marine mammal (e.g., a whale, a
seal, an elephant seal, a dolphin, a sea lion; etc.) and the like.
Any type of cell may be of interest (e.g. a stem cell, e.g. an
embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a
germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia,
etc.), an adult stem cell, a somatic cell, e.g. a fibroblast, a
hematopoietic cell, a neuron, a muscle cell, a bone cell, a
hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic
cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell,
8-cell, etc. stage zebrafish embryo; etc.).
[0376] Cells may be from established cell lines or they may be
primary cells, where "primary cells", "primary cell lines", and
"primary cultures" are used interchangeably herein to refer to
cells and cells cultures that have been derived from a subject and
allowed to grow in vitro for a limited number of passages, i.e.
splittings, of the culture. For example, primary cultures are
cultures that may have been passaged 0 times, 1 time, 2 times, 4
times, 5 times, 10 times, or 15 times, but not enough times go
through the crisis stage. Typically, the primary cell lines are
maintained for fewer than 10 passages in vitro. Target cells can be
unicellular organisms and/or can be grown in culture. If the cells
are primary cells, they may be harvest from an individual by any
convenient method. For example, leukocytes may be conveniently
harvested by apheresis, leukocytapheresis, density gradient
separation, etc., while cells from tissues such as skin, muscle,
bone marrow, spleen, liver, pancreas, lung, intestine, stomach,
etc. can be conveniently harvested by biopsy.
[0377] In some of the above applications, the subject methods may
be employed to induce target nucleic acid cleavage, target nucleic
acid modification, and/or to bind target nucleic acids (e.g., for
visualization, for collecting and/or analyzing, etc.) in mitotic or
post-mitotic cells in vivo and/or ex vivo and/or in vitro (e.g., to
disrupt production of a protein encoded by a targeted mRNA, to
cleave or otherwise modify target DNA, to genetically modify a
target cell, and the like). Because the guide RNA provides
specificity by hybridizing to target nucleic acid, a mitotic and/or
post-mitotic cell of interest in the disclosed methods may include
a cell from any organism (e.g. a bacterial cell, an archaeal cell,
a cell of a single-cell eukaryotic organism, a plant cell, an algal
cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii,
Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens,
C. agardh, and the like, a fungal cell (e.g., a yeast cell), an
animal cell, a cell from an invertebrate animal (e.g. fruit fly,
cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate
animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from
a mammal, a cell from a rodent, a cell from a human, etc.). In some
cases, a subject Cas12J protein (and/or nucleic acid encoding the
protein such as DNA and/or RNA), and/or Cas12J guide RNA (and/or a
DNA encoding the guide RNA), and/or donor template, and/or RNP can
be introduced into an individual (i.e., the target cell can be in
vivo) (e.g., a mammal, a rat, a mouse, a pig, a primate, a
non-human primate, a human, etc.). In some case, such an
administration can be for the purpose of treating and/or preventing
a disease, e.g., by editing the genome of targeted cells.
[0378] Plant cells include cells of a monocotyledon, and cells of a
dicotyledon. The cells can be root cells, leaf cells, cells of the
xylem, cells of the phloem, cells of the cambium, apical meristem
cells, parenchyma cells, collenchyma cells, sclerenchyma cells, and
the like. Plant cells include cells of agricultural crops such as
wheat, corn, rice, sorghum, millet, soybean, etc. Plant cells
include cells of agricultural fruit and nut plants, e.g., plant
that produce apricots, oranges, lemons, apples, plums, pears,
almonds, etc.
[0379] Additional examples of target cells are listed above in the
section titled "Modified cells." Non-limiting examples of cells
(target cells) include: a prokaryotic cell, eukaryotic cell, a
bacterial cell, an archaeal cell, a cell of a single-cell
eukaryotic organism, a protozoa cell, a cell from a plant (e.g.,
cells from plant crops, fruits, vegetables, grains, soy bean, corn,
maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin,
hay, potatos, cotton, cannabis, tobacco, flowering plants,
conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts,
liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal
cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii,
Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens,
C. agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g.,
a yeast cell, a cell from a mushroom), an animal cell, a cell from
an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm,
nematode, etc.), a cell from a vertebrate animal (e.g., fish,
amphibian, reptile, bird, mammal), a cell from a mammal (e.g., an
ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a
rat, a mouse); a non-human primate; a human; a feline (e.g., a
cat); a canine (e.g., a dog); etc.), and the like. In some cases,
the cell is a cell that does not originate from a natural organism
(e.g., the cell can be a synthetically made cell; also referred to
as an artificial cell).
[0380] A cell can be an in vitro cell (e.g., established cultured
cell line). A cell can be an ex vivo cell (cultured cell from an
individual). A cell can be and in vivo cell (e.g., a cell in an
individual). A cell can be an isolated cell. A cell can be a cell
inside of an organism. A cell can be an organism. A cell can be a
cell in a cell culture (e.g., in vitro cell culture). A cell can be
one of a collection of cells. A cell can be a prokaryotic cell or
derived from a prokaryotic cell. A cell can be a bacterial cell or
can be derived from a bacterial cell. A cell can be an archaeal
cell or derived from an archaeal cell. A cell can be a eukaryotic
cell or derived from a eukaryotic cell. A cell can be a plant cell
or derived from a plant cell. A cell can be an animal cell or
derived from an animal cell. A cell can be an invertebrate cell or
derived from an invertebrate cell. A cell can be a vertebrate cell
or derived from a vertebrate cell. A cell can be a mammalian cell
or derived from a mammalian cell. A cell can be a rodent cell or
derived from a rodent cell. A cell can be a human cell or derived
from a human cell. A cell can be a microbe cell or derived from a
microbe cell. A cell can be a fungi cell or derived from a fungi
cell. A cell can be an insect cell. A cell can be an arthropod
cell. A cell can be a protozoan cell. A cell can be a helminth
cell.
[0381] Suitable cells include a stem cell (e.g. an embryonic stem
(ES) cell, an induced pluripotent stem (iPS) cell; a germ cell
(e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a
somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell,
a hematopoietic cell, a neuron, a muscle cell, a bone cell, a
hepatocyte, a pancreatic cell, etc.
[0382] Suitable cells include human embryonic stem cells, fetal
cardiomyocytes, myofibroblasts, mesenchymal stem cells,
cardiomyocytes, adipocytes, totipotent cells, pluripotent cells,
blood stem cells, myoblasts, adult stem cells, bone marrow cells,
mesenchymal cells, embryonic stem cells, parenchymal cells,
epithelial cells, endothelial cells, mesothelial cells,
fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous
cells, stem cells, hematopoietic stem cells, bone-marrow derived
progenitor cells, myocardial cells, skeletal cells, fetal cells,
undifferentiated cells, multi-potent progenitor cells, unipotent
progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts,
macrophages, capillary endothelial cells, xenogenic cells,
allogenic cells, and post-natal stem cells.
[0383] In some cases, the cell is an immune cell, a neuron, an
epithelial cell, and endothelial cell, or a stem cell. In some
cases, the immune cell is a T cell, a B cell, a monocyte, a natural
killer cell, a dendritic cell, or a macrophage. In some cases, the
immune cell is a cytotoxic T cell. In some cases, the immune cell
is a helper T cell. In some cases, the immune cell is a regulatory
T cell (Treg).
[0384] In some cases, the cell is a stem cell. Stem cells include
adult stem cells. Adult stem cells are also referred to as somatic
stem cells.
[0385] Adult stem cells are resident in differentiated tissue, but
retain the properties of self-renewal and ability to give rise to
multiple cell types, usually cell types typical of the tissue in
which the stem cells are found. Numerous examples of somatic stem
cells are known to those of skill in the art, including muscle stem
cells; hematopoietic stem cells; epithelial stem cells; neural stem
cells; mesenchymal stem cells; mammary stem cells; intestinal stem
cells; mesodermal stem cells; endothelial stem cells; olfactory
stem cells; neural crest stem cells; and the like.
[0386] Stem cells of interest include mammalian stem cells, where
the term "mammalian" refers to any animal classified as a mammal,
including humans; non-human primates; domestic and farm animals;
and zoo, laboratory, sports, or pet animals, such as dogs, horses,
cats, cows, mice, rats, rabbits, etc. In some cases, the stem cell
is a human stem cell. In some cases, the stem cell is a rodent
(e.g., a mouse; a rat) stem cell. In some cases, the stem cell is a
non-human primate stem cell.
[0387] Stem cells can express one or more stem cell markers, e.g.,
SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1,
OLFM4, CDH17, and PPARGC1A.
[0388] In some cases, the stem cell is a hematopoietic stem cell
(HSC). HSCs are mesoderm-derived cells that can be isolated from
bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are
characterized as CD34.sup.+ and CD3.sup.-. HSCs can repopulate the
erythroid, neutrophil-macrophage, megakaryocyte and lymphoid
hematopoietic cell lineages in vivo. In vitro, HSCs can be induced
to undergo at least some self-renewing cell divisions and can be
induced to differentiate to the same lineages as is seen in vivo.
As such, HSCs can be induced to differentiate into one or more of
erythroid cells, megakaryocytes, neutrophils, macrophages, and
lymphoid cells.
[0389] In other embodiments, the stem cell is a neural stem cell
(NSC). Neural stem cells (NSCs) are capable of differentiating into
neurons, and glia (including oligodendrocytes, and astrocytes). A
neural stem cell is a multipotent stem cell which is capable of
multiple divisions, and under specific conditions can produce
daughter cells which are neural stem cells, or neural progenitor
cells that can be neuroblasts or glioblasts, e.g., cells committed
to become one or more types of neurons and glial cells
respectively. Methods of obtaining NSCs are known in the art.
[0390] In other embodiments, the stem cell is a mesenchymal stem
cell (MSC). MSCs originally derived from the embryonal mesoderm and
isolated from adult bone marrow, can differentiate to form muscle,
bone, cartilage, fat, marrow stroma, and tendon. Methods of
isolating MSC are known in the art; and any known method can be
used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which
describes isolation of human MSC.
[0391] A cell is in some cases a plant cell. A plant cell can be a
cell of a monocotyledon. A cell can be a cell of a dicotyledon.
[0392] In some cases, the cell is a plant cell. For example, the
cell can be a cell of a major agricultural plant, e.g., Barley,
Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland),
Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice,
Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil),
Sunflowers (Non-Oil), Sweet Potatoes, Tobacco (Burley), Tobacco
(Flue-cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat
(Winter), and the like. As another example, the cell is a cell of a
vegetable crops which include but are not limited to, e.g., alfalfa
sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus,
bamboo shoots, banana flowers, bean sprouts, beans, beet tops,
beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini),
brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales),
calabaza, cardoon, carrots, cauliflower, celery, chayote, chinese
artichoke (crosnes), chinese cabbage, chinese celery, chinese
chives, choy sum, chrysanthemum leaves (tung ho), collard greens,
corn stalks, corn-sweet, cucumbers, daikon, dandelion greens,
dasheen, dau mue (pea tips), donqua (winter melon), eggplant,
endive, escarole, fiddle head ferns, field cress, frisee, gai choy
(chinese mustard), gailon, galanga (siam, thai ginger), garlic,
ginger root, gobo, greens, hanover salad greens, huauzontle,
jerusalem artichokes, jicama, kale greens, kohlrabi, lamb's
quarters (quilete), lettuce (bibb), lettuce (boston), lettuce
(boston red), lettuce (green leaf), lettuce (iceberg), lettuce
(lolla rossa), lettuce (oak leaf--green), lettuce (oak leaf--red),
lettuce (processed), lettuce (red leaf), lettuce (romaine), lettuce
(ruby romaine), lettuce (russian red mustard), linkok, lo bok, long
beans, lotus root, mache, maguey (agave) leaves, malanga, mesculin
mix, mizuna, moap (smooth luffa), moo, moqua (fuzzy squash),
mushrooms, mustard, nagaimo, okra, ong choy, onions green, opo
(long squash), ornamental corn, ornamental gourds, parsley,
parsnips, peas, peppers (bell type), peppers, pumpkins, radicchio,
radish sprouts, radishes, rape greens, rape greens, rhubarb,
romaine (baby red), rutabagas, salicornia (sea bean), sinqua
(angled/ridged luffa), spinach, squash, straw bales, sugarcane,
sweet potatoes, swiss chard, tamarindo, taro, taro leaf, taro
shoots, tatsoi, tepeguaje (guaje), tindora, tomatillos, tomatoes,
tomatoes (cherry), tomatoes (grape type), tomatoes (plum type),
tumeric, turnip tops greens, turnips, water chestnuts, yampi, yams,
yu choy, yuca (cassava), and the like.
[0393] A cell is in some cases an arthropod cell. For example, the
cell can be a cell of a sub-order, a family, a sub-family, a group,
a sub-group, or a species of, e.g., Chelicerata, Myriapodia,
Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura,
Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera,
Neoptera, Exopterygota, Plecoptera, Embioptera, Orthoptera,
Zoraptera, Dermaptera, Dictyoptera, Notoptera, Grylloblattidae,
Mantophasmatidae, Phasmatodea, Blattaria, Isoptera, Mantodea,
Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera,
Endopterygota or Holometabola, Hymenoptera, Coleoptera,
Strepsiptera, Raphidioptera, Megaloptera, Neuroptera, Mecoptera,
Siphonaptera, Diptera, Trichoptera, or Lepidoptera.
[0394] A cell is in some cases an insect cell. For example, in some
cases, the cell is a cell of a mosquito, a grasshopper, a true bug,
a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a
beetle.
Introducing Components into a Target Cell
[0395] A Cas12J guide RNA (or a nucleic acid comprising a
nucleotide sequence encoding same), and/or a Cas12J fusion
polypeptide (or a nucleic acid comprising a nucleotide sequence
encoding same) and/or a donor polynucleotide can be introduced into
a host cell by any of a variety of well-known methods.
[0396] Methods of introducing a nucleic acid into a cell are known
in the art, and any convenient method can be used to introduce a
nucleic acid (e.g., an expression construct) into a taret cell
(e.g., eukaryotic cell, human cell, stem cell, progenitor cell, and
the like). Suitable methods are described in more detail elsewhere
herein and include e.g., viral or bacteriophage infection,
transfection, conjugation, protoplast fusion, lipofection,
electroporation, calcium phosphate precipitation, polyethyleneimine
(PEI)-mediated transfection, DEAE-dextran mediated transfection,
liposome-mediated transfection, particle gun technology, calcium
phosphate precipitation, direct micro injection,
nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et.,
al Adv Drug Deliv Rev. 2012 Sep. 13. pii: S0169-409X(12)00283-9.
doi: 10.1016/j.addr.2012.09.023), and the like. Any or all of the
components can be introduced into a cell as a composition (e.g.,
including any convenient combination of: a a Cas12J polypeptide, a
Cas12J guide RNA, a donor polynucleotide, etc.) using known
methods, e.g., such as nucleofection.
Donor Polynucleotide (Donor Template)
[0397] Guided by a Cas12J guide RNA, a Cas12J protein in some cases
generates site-specific double strand breaks (DSBs) or single
strand breaks (SSBs) (e.g., when the Cas12J protein is a nickase
variant) within double-stranded DNA (dsDNA) target nucleic acids,
which are repaired either by non-homologous end joining (NHEJ) or
homology-directed recombination (HDR).
[0398] In some cases, contacting a target DNA (with a Cas12J
protein and a Cas12J guide RNA) occurs under conditions that are
permissive for nonhomologous end joining or homology-directed
repair. Thus, in some cases, a subject method includes contacting
the target DNA with a donor polynucleotide (e.g., by introducing
the donor polynucleotide into a cell), wherein the donor
polynucleotide, a portion of the donor polynucleotide, a copy of
the donor polynucleotide, or a portion of a copy of the donor
polynucleotide integrates into the target DNA. In some cases, the
method does not comprise contacting a cell with a donor
polynucleotide, and the target DNA is modified such that
nucleotides within the target DNA are deleted.
[0399] In some cases, Cas12J guide RNA (or DNA encoding same) and a
Cas12J protein (or a nucleic acid encoding same, such as an RNA or
a DNA, e.g., one or more expression vectors) are coadministered
(e.g., contacted with a target nucleic acid, administered to cells,
etc.) with a donor polynucleotide sequence that includes at least a
segment with homology to the target DNA sequence, the subject
methods may be used to add, i.e. insert or replace, nucleic acid
material to a target DNA sequence (e.g. to "knock in" a nucleic
acid, e.g., one that encodes for a protein, an siRNA, an miRNA,
etc.), to add a tag (e.g., 6.times.His, a fluorescent protein
(e.g., a green fluorescent protein; a yellow fluorescent protein,
etc.), hemagglutinin (HA), FLAG, etc.), to add a regulatory
sequence to a gene (e.g. promoter, polyadenylation signal, internal
ribosome entry sequence (IRES), 2A peptide, start codon, stop
codon, splice signal, localization signal, etc.), to modify a
nucleic acid sequence (e.g., introduce a mutation, remove a disease
causing mutation by introducing a correct sequence), and the like.
As such, a complex comprising a Cas12J guide RNA and Cas12J protein
is useful in any in vitro or in vivo application in which it is
desirable to modify DNA in a site-specific, i.e. "targeted", way,
for example gene knock-out, gene knock-in, gene editing, gene
tagging, etc., as used in, for example, gene therapy, e.g. to treat
a disease or as an antiviral, antipathogenic, or anticancer
therapeutic, the production of genetically modified organisms in
agriculture, the large scale production of proteins by cells for
therapeutic, diagnostic, or research purposes, the induction of iPS
cells, biological research, the targeting of genes of pathogens for
deletion or replacement, etc.
[0400] In applications in which it is desirable to insert a
polynucleotide sequence into the genome where a target sequence is
cleaved, a donor polynucleotide (a nucleic acid comprising a donor
sequence) can also be provided to the cell. By a "donor sequence"
or "donor polynucleotide" or "donor template" it is meant a nucleic
acid sequence to be inserted at the site cleaved by the Cas12J
protein (e.g., after dsDNA cleavage, after nicking a target DNA,
after dual nicking a target DNA, and the like). The donor
polynucleotide can contain sufficient homology to a genomic
sequence at the target site, e.g. 70%, 80%, 85%, 90%, 95%, or 100%
homology with the nucleotide sequences flanking the target site,
e.g. within about 50 bases or less of the target site, e.g. within
about 30 bases, within about 15 bases, within about 10 bases,
within about 5 bases, or immediately flanking the target site, to
support homology-directed repair between it and the genomic
sequence to which it bears homology. Approximately 25, 50, 100, or
200 nucleotides, or more than 200 nucleotides, of sequence homology
between a donor and a genomic sequence (or any integral value
between 10 and 200 nucleotides, or more) can support
homology-directed repair. Donor polynucleotides can be of any
length, e.g. 10 nucleotides or more, 50 nucleotides or more, 100
nucleotides or more, 250 nucleotides or more, 500 nucleotides or
more, 1000 nucleotides or more, 5000 nucleotides or more, etc.
[0401] The donor sequence is typically not identical to the genomic
sequence that it replaces. Rather, the donor sequence may contain
at least one or more single base changes, insertions, deletions,
inversions or rearrangements with respect to the genomic sequence,
so long as sufficient homology is present to support
homology-directed repair (e.g., for gene correction, e.g., to
convert a disease-causing base pair to a non disease-causing base
pair). In some embodiments, the donor sequence comprises a
non-homologous sequence flanked by two regions of homology, such
that homology-directed repair between the target DNA region and the
two flanking sequences results in insertion of the non-homologous
sequence at the target region. Donor sequences may also comprise a
vector backbone containing sequences that are not homologous to the
DNA region of interest and that are not intended for insertion into
the DNA region of interest. Generally, the homologous region(s) of
a donor sequence will have at least 50% sequence identity to a
genomic sequence with which recombination is desired. In certain
embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence
identity is present. Any value between 1% and 100% sequence
identity can be present, depending upon the length of the donor
polynucleotide.
[0402] The donor sequence may comprise certain sequence differences
as compared to the genomic sequence, e.g. restriction sites,
nucleotide polymorphisms, selectable markers (e.g., drug resistance
genes, fluorescent proteins, enzymes etc.), etc., which may be used
to assess for successful insertion of the donor sequence at the
cleavage site or in some cases may be used for other purposes
(e.g., to signify expression at the targeted genomic locus). In
some cases, if located in a coding region, such nucleotide sequence
differences will not change the amino acid sequence, or will make
silent amino acid changes (i.e., changes which do not affect the
structure or function of the protein). Alternatively, these
sequences differences may include flanking recombination sequences
such as FLPs, loxP sequences, or the like, that can be activated at
a later time for removal of the marker sequence.
[0403] In some cases, the donor sequence is provided to the cell as
single-stranded DNA. In some cases, the donor sequence is provided
to the cell as double-stranded DNA. It may be introduced into a
cell in linear or circular form. If introduced in linear form, the
ends of the donor sequence may be protected (e.g., from
exonucleolytic degradation) by any convenient method and such
methods are known to those of skill in the art. For example, one or
more dideoxynucleotide residues can be added to the 3' terminus of
a linear molecule and/or self-complementary oligonucleotides can be
ligated to one or both ends. See, for example, Chang et al. (1987)
Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science
272:886-889. Additional methods for protecting exogenous
polynucleotides from degradation include, but are not limited to,
addition of terminal amino group(s) and the use of modified
internucleotide linkages such as, for example, phosphorothioates,
phosphoramidates, and O-methyl ribose or deoxyribose residues. As
an alternative to protecting the termini of a linear donor
sequence, additional lengths of sequence may be included outside of
the regions of homology that can be degraded without impacting
recombination. A donor sequence can be introduced into a cell as
part of a vector molecule having additional sequences such as, for
example, replication origins, promoters and genes encoding
antibiotic resistance. Moreover, donor sequences can be introduced
as naked nucleic acid, as nucleic acid complexed with an agent such
as a liposome or poloxamer, or can be delivered by viruses (e.g.,
adenovirus, AAV), as described elsewhere herein for nucleic acids
encoding a Cas12J guide RNA and/or a Cas12J fusion polypeptide
and/or donor polynucleotide.
Detection Methods
[0404] A Cas12J polypeptide of the present disclosure can
promiscuously cleave non-targeted single stranded DNA (ssDNA) once
activated by detection of a target DNA (double or single stranded).
Once a Cas12J polypeptide of the present disclosure is activated by
a guide RNA, which occurs when the guide RNA hybridizes to a target
sequence of a target DNA (i.e., the sample includes the targeted
DNA), the Cas12J polypeptide becomes a nuclease that promiscuously
cleaves ssDNAs (i.e., the nuclease cleaves non-target ssDNAs, i.e.,
ssDNAs to which the guide sequence of the guide RNA does not
hybridize). Thus, when the target DNA is present in the sample
(e.g., in some cases above a threshold amount), the result is
cleavage of ssDNAs in the sample, which can be detected using any
convenient detection method (e.g., using a labeled single stranded
detector DNA). Cleavage of non-target nucleic acid is referred to
as "trans cleavage." In some cases, a Cas12J effector polypeptide
of the present disclosure mediates trans cleavage of ssDNA, but not
ssRNA.
[0405] Provided are compositions and methods for detecting a target
DNA (double stranded or single stranded) in a sample. In some
cases, a detector DNA is used that is single stranded (ssDNA) and
does not hybridize with the guide sequence of the guide RNA (i.e.,
the detector ssDNA is a non-target ssDNA). Such methods can include
(a) contacting the sample with: (i) a Cas12J polypeptide of the
present disclosure; (ii) a guide RNA comprising: a region that
binds to the Cas12J polypeptide, and a guide sequence that
hybridizes with the target DNA; and (iii) a detector DNA that is
single stranded and does not hybridize with the guide sequence of
the guide RNA; and (b) measuring a detectable signal produced by
cleavage of the single stranded detector DNA by the Cas12J
polypeptide, thereby detecting the target DNA. As noted above, once
a Cas12J polypeptide of the present disclosure is activated by a
guide RNA, which occurs when the sample includes a target DNA to
which the guide RNA hybridizes (i.e., the sample includes the
targeted target DNA), the Cas12J polypeptide is activated and
functions as an endoribonuclease that non-specifically cleaves
ssDNAs (including non-target ssDNAs) present in the sample. Thus,
when the targeted target DNA is present in the sample (e.g., in
some cases above a threshold amount), the result is cleavage of
ssDNA (including non-target ssDNA) in the sample, which can be
detected using any convenient detection method (e.g., using a
labeled detector ssDNA).
[0406] Also provided are compositions and methods for cleaving
single stranded DNAs (ssDNAs) (e.g., non-target ssDNAs). Such
methods can include contacting a population of nucleic acids,
wherein said population comprises a target DNA and a plurality of
non-target ssDNAs, with: (i) a Cas12J polypeptide of the present
disclosure; and (ii) a guide RNA comprising: a region that binds to
the Cas12J polypeptide and a guide sequence that hybridizes with
the target DNA, wherein the Cas12J polypeptide cleaves non-target
ssDNAs of said plurality. Such a method can be used, e.g., to
cleave foreign ssDNAs (e.g., viral DNAs) in a cell.
[0407] The contacting step of a subject method can be carried out
in a composition comprising divalent metal ions. The contacting
step can be carried out in an acellular environment, e.g., outside
of a cell. The contacting step can be carried out inside a cell.
The contacting step can be carried out in a cell in vitro. The
contacting step can be carried out in a cell ex vivo. The
contacting step can be carried out in a cell in vivo.
[0408] The guide RNA can be provided as RNA or as a nucleic acid
encoding the guide RNA (e.g., a DNA such as a recombinant
expression vector). The Cas12J polypeptide can be provided as a
protein or as a nucleic acid encoding the protein (e.g., an mRNA, a
DNA such as a recombinant expression vector). In some cases, two or
more (e.g., 3 or more, 4 or more, 5 or more, or 6 or more) guide
RNAs can be provided by (e.g., using a precursor guide RNA array,
which can be cleaved by the Cas12J effector protein into individual
("mature") guide RNAs).
[0409] In some cases (e.g., when contacting with a guide RNA and a
Cas12J polypeptide of the present disclosure, the sample is
contacted for 2 hours or less (e.g., 1.5 hours or less, 1 hour or
less, 40 minutes or less, 30 minutes or less, 20 minutes or less,
10 minutes or less, or 5 minutes or less, or 1 minute or less)
prior to the measuring step. For example, in some cases the sample
is contacted for 40 minutes or less prior to the measuring step. In
some cases, the sample is contacted for 20 minutes or less prior to
the measuring step. In some cases, the sample is contacted for 10
minutes or less prior to the measuring step. In some cases, the
sample is contacted for 5 minutes or less prior to the measuring
step. In some cases, the sample is contacted for 1 minute or less
prior to the measuring step. In some cases, the sample is contacted
for from 50 seconds to 60 seconds prior to the measuring step. In
some cases, the sample is contacted for from 40 seconds to 50
seconds prior to the measuring step. In some cases, the sample is
contacted for from 30 seconds to 40 seconds prior to the measuring
step. In some cases, the sample is contacted for from 20 seconds to
30 seconds prior to the measuring step. In some cases, the sample
is contacted for from 10 seconds to 20 seconds prior to the
measuring step.
[0410] A method of the present disclosure for detecting a target
DNA (single-stranded or double-stranded) in a sample can detect a
target DNA with a high degree of sensitivity. In some cases, a
method of the present disclosure can be used to detect a target DNA
present in a sample comprising a plurality of DNAs (including the
target DNA and a plurality of non-target DNAs), where the target
DNA is present at one or more copies per 10.sup.7 non-target DNAs
(e.g., one or more copies per 10.sup.6 non-target DNAs, one or more
copies per 10.sup.5 non-target DNAs, one or more copies per
10.sup.4 non-target DNAs, one or more copies per 10.sup.3
non-target DNAs, one or more copies per 10.sup.2 non-target DNAs,
one or more copies per 50 non-target DNAs, one or more copies per
20 non-target DNAs, one or more copies per 10 non-target DNAs, or
one or more copies per 5 non-target DNAs). In some cases, a method
of the present disclosure can be used to detect a target DNA
present in a sample comprising a plurality of DNAs (including the
target DNA and a plurality of non-target DNAs), where the target
DNA is present at one or more copies per 10.sup.18 non-target DNAs
(e.g., one or more copies per 10.sup.15 non-target DNAs, one or
more copies per 10.sup.12 non-target DNAs, one or more copies per
10.sup.9 non-target DNAs, one or more copies per 10.sup.6
non-target DNAs, one or more copies per 10.sup.5 non-target DNAs,
one or more copies per 10.sup.4 non-target DNAs, one or more copies
per 10.sup.3 non-target DNAs, one or more copies per 10.sup.2
non-target DNAs, one or more copies per 50 non-target DNAs, one or
more copies per 20 non-target DNAs, one or more copies per 10
non-target DNAs, or one or more copies per 5 non-target DNAs).
[0411] In some cases, a method of the present disclosure can detect
a target DNA present in a sample, where the target DNA is present
at from one copy per 10.sup.7 non-target DNAs to one copy per 10
non-target DNAs (e.g., from 1 copy per 10.sup.7 non-target DNAs to
1 copy per 10.sup.2 non-target DNAs, from 1 copy per 10.sup.7
non-target DNAs to 1 copy per 10.sup.3 non-target DNAs, from 1 copy
per 10.sup.7 non-target DNAs to 1 copy per 10.sup.4 non-target
DNAs, from 1 copy per 10.sup.7 non-target DNAs to 1 copy per
10.sup.5 non-target DNAs, from 1 copy per 10.sup.7 non-target DNAs
to 1 copy per 10.sup.6 non-target DNAs, from 1 copy per 10.sup.6
non-target DNAs to 1 copy per 10 non-target DNAs, from 1 copy per
10.sup.6 non-target DNAs to 1 copy per 10.sup.2 non-target DNAs,
from 1 copy per 10.sup.6 non-target DNAs to 1 copy per 10.sup.3
non-target DNAs, from 1 copy per 10.sup.6 non-target DNAs to 1 copy
per 10.sup.4 non-target DNAs, from 1 copy per 10.sup.6 non-target
DNAs to 1 copy per 10.sup.5 non-target DNAs, from 1 copy per
10.sup.5 non-target DNAs to 1 copy per 10 non-target DNAs, from 1
copy per 10.sup.5 non-target DNAs to 1 copy per 10.sup.2 non-target
DNAs, from 1 copy per 10.sup.5 non-target DNAs to 1 copy per
10.sup.3 non-target DNAs, or from 1 copy per 10.sup.5 non-target
DNAs to 1 copy per 10.sup.4 non-target DNAs).
[0412] In some cases, a method of the present disclosure can detect
a target DNA present in a sample, where the target DNA is present
at from one copy per 10.sup.18 non-target DNAs to one copy per 10
non-target DNAs (e.g., from 1 copy per 10.sup.18 non-target DNAs to
1 copy per 10.sup.2 non-target DNAs, from 1 copy per 10.sup.15
non-target DNAs to 1 copy per 10.sup.2 non-target DNAs, from 1 copy
per 10.sup.12 non-target DNAs to 1 copy per 10.sup.2 non-target
DNAs, from 1 copy per 10.sup.9 non-target DNAs to 1 copy per
10.sup.2 non-target DNAs, from 1 copy per 10.sup.7 non-target DNAs
to 1 copy per 10.sup.2 non-target DNAs, from 1 copy per 10.sup.2
non-target DNAs to 1 copy per 10.sup.3 non-target DNAs, from 1 copy
per 10.sup.7 non-target DNAs to 1 copy per 10.sup.4 non-target
DNAs, from 1 copy per 10.sup.7 non-target DNAs to 1 copy per
10.sup.5 non-target DNAs, from 1 copy per 10.sup.7 non-target DNAs
to 1 copy per 10.sup.6 non-target DNAs, from 1 copy per 10.sup.6
non-target DNAs to 1 copy per 10 non-target DNAs, from 1 copy per
10.sup.6 non-target DNAs to 1 copy per 10.sup.2 non-target DNAs,
from 1 copy per 10.sup.6 non-target DNAs to 1 copy per 10.sup.3
non-target DNAs, from 1 copy per 10.sup.6 non-target DNAs to 1 copy
per 10.sup.4 non-target DNAs, from 1 copy per 10.sup.6 non-target
DNAs to 1 copy per 10.sup.5 non-target DNAs, from 1 copy per
10.sup.5 non-target DNAs to 1 copy per 10 non-target DNAs, from 1
copy per 10.sup.5 non-target DNAs to 1 copy per 10.sup.2 non-target
DNAs, from 1 copy per 10.sup.5 non-target DNAs to 1 copy per
10.sup.3 non-target DNAs, or from 1 copy per 10.sup.5 non-target
DNAs to 1 copy per 10.sup.4 non-target DNAs).
[0413] In some cases, a method of the present disclosure can detect
a target DNA present in a sample, where the target DNA is present
at from one copy per 10.sup.7 non-target DNAs to one copy per 100
non-target DNAs (e.g., from 1 copy per 10.sup.7 non-target DNAs to
1 copy per 10.sup.2 non-target DNAs, from 1 copy per 10.sup.7
non-target DNAs to 1 copy per 10.sup.3 non-target DNAs, from 1 copy
per 10.sup.7 non-target DNAs to 1 copy per 10.sup.4 non-target
DNAs, from 1 copy per 10.sup.7 non-target DNAs to 1 copy per
10.sup.5 non-target DNAs, from 1 copy per 10.sup.7 non-target DNAs
to 1 copy per 10.sup.6 non-target DNAs, from 1 copy per 10.sup.6
non-target DNAs to 1 copy per 100 non-target DNAs, from 1 copy per
10.sup.6 non-target DNAs to 1 copy per 100 non-target DNAs, from 1
copy per 10.sup.6 non-target DNAs to 1 copy per 10.sup.3 non-target
DNAs, from 1 copy per 10.sup.6 non-target DNAs to 1 copy per 10
non-target DNAs, from 1 copy per 10.sup.6 non-target DNAs to 1 copy
per 10.sup.5 non-target DNAs, from 1 copy per 10.sup.5 non-target
DNAs to 1 copy per 100 non-target DNAs, from 1 copy per 10.sup.5
non-target DNAs to 1 copy per 10.sup.2 non-target DNAs, from 1 copy
per 10.sup.5 non-target DNAs to 1 copy per 10.sup.3 non-target
DNAs, or from 1 copy per 10.sup.5 non-target DNAs to 1 copy per
10.sup.4 non-target DNAs).
[0414] In some cases, the threshold of detection, for a subject
method of detecting a target DNA in a sample, is 10 nM or less. The
term "threshold of detection" is used herein to describe the
minimal amount of target DNA that must be present in a sample in
order for detection to occur. Thus, as an illustrative example,
when a threshold of detection is 10 nM, then a signal can be
detected when a target DNA is present in the sample at a
concentration of 10 nM or more. In some cases, a method of the
present disclosure has a threshold of detection of 5 nM or less. In
some cases, a method of the present disclosure has a threshold of
detection of 1 nM or less. In some cases, a method of the present
disclosure has a threshold of detection of 0.5 nM or less. In some
cases, a method of the present disclosure has a threshold of
detection of 0.1 nM or less. In some cases, a method of the present
disclosure has a threshold of detection of 0.05 nM or less. In some
cases, a method of the present disclosure has a threshold of
detection of 0.01 nM or less. In some cases, a method of the
present disclosure has a threshold of detection of 0.005 nM or
less. In some cases, a method of the present disclosure has a
threshold of detection of 0.001 nM or less. In some cases, a method
of the present disclosure has a threshold of detection of 0.0005 nM
or less. In some cases, a method of the present disclosure has a
threshold of detection of 0.0001 nM or less. In some cases, a
method of the present disclosure has a threshold of detection of
0.00005 nM or less. In some cases, a method of the present
disclosure has a threshold of detection of 0.00001 nM or less. In
some cases, a method of the present disclosure has a threshold of
detection of 10 pM or less. In some cases, a method of the present
disclosure has a threshold of detection of 1 pM or less. In some
cases, a method of the present disclosure has a threshold of
detection of 500 fM or less. In some cases, a method of the present
disclosure has a threshold of detection of 250 fM or less. In some
cases, a method of the present disclosure has a threshold of
detection of 100 fM or less. In some cases, a method of the present
disclosure has a threshold of detection of 50 fM or less. In some
cases, a method of the present disclosure has a threshold of
detection of 500 aM (attomolar) or less. In some cases, a method of
the present disclosure has a threshold of detection of 250 aM or
less. In some cases, a method of the present disclosure has a
threshold of detection of 100 aM or less. In some cases, a method
of the present disclosure has a threshold of detection of 50 aM or
less. In some cases, a method of the present disclosure has a
threshold of detection of 10 aM or less. In some cases, a method of
the present disclosure has a threshold of detection of 1 aM or
less.
[0415] In some cases, the threshold of detection (for detecting the
target DNA in a subject method), is in a range of from 500 fM to 1
nM (e.g., from 500 fM to 500 pM, from 500 fM to 200 pM, from 500 fM
to 100 pM, from 500 fM to 10 pM, from 500 fM to 1 pM, from 800 fM
to 1 nM, from 800 fM to 500 pM, from 800 fM to 200 pM, from 800 fM
to 100 pM, from 800 fM to 10 pM, from 800 fM to 1 pM, from 1 pM to
1 nM, from 1 pM to 500 pM, from 1 pM to 200 pM, from 1 pM to 100
pM, or from 1 pM to 10 pM) (where the concentration refers to the
threshold concentration of target DNA at which the target DNA can
be detected). In some cases, a method of the present disclosure has
a threshold of detection in a range of from 800 fM to 100 pM. In
some cases, a method of the present disclosure has a threshold of
detection in a range of from 1 pM to 10 pM. In some cases, a method
of the present disclosure has a threshold of detection in a range
of from 10 fM to 500 fM, e.g., from 10 fM to 50 fM, from 50 fM to
100 fM, from 100 fM to 250 fM, or from 250 fM to 500 fM.
[0416] In some cases, the minimum concentration at which a target
DNA can be detected in a sample is in a range of from 500 fM to 1
nM (e.g., from 500 fM to 500 pM, from 500 fM to 200 pM, from 500 fM
to 100 pM, from 500 fM to 10 pM, from 500 fM to 1 pM, from 800 fM
to 1 nM, from 800 fM to 500 pM, from 800 fM to 200 pM, from 800 fM
to 100 pM, from 800 fM to 10 pM, from 800 fM to 1 pM, from 1 pM to
1 nM, from 1 pM to 500 pM, from 1 pM to 200 pM, from 1 pM to 100
pM, or from 1 pM to 10 pM). In some cases, the minimum
concentration at which a target DNA can be detected in a sample is
in a range of from 800 fM to 100 pM. In some cases, the minimum
concentration at which a target DNA can be detected in a sample is
in a range of from 1 pM to 10 pM.
[0417] In some cases, the threshold of detection (for detecting the
target DNA in a subject method), is in a range of from 1 aM to 1 nM
(e.g., from 1 aM to 500 pM, from 1 aM to 200 pM, from 1 aM to 100
pM, from 1 aM to 10 pM, from 1 aM to 1 pM, from 100 aM to 1 nM,
from 100 aM to 500 pM, from 100 aM to 200 pM, from 100 aM to 100
pM, from 100 aM to 10 pM, from 100 aM to 1 pM, from 250 aM to 1 nM,
from 250 aM to 500 pM, from 250 aM to 200 pM, from 250 aM to 100
pM, from 250 aM to 10 pM, from 250 aM to 1 pM, from 500 aM to 1 nM,
from 500 aM to 500 pM, from 500 aM to 200 pM, from 500 aM to 100
pM, from 500 aM to 10 pM, from 500 aM to 1 pM, from 750 aM to 1 nM,
from 750 aM to 500 pM, from 750 aM to 200 pM, from 750 aM to 100
pM, from 750 aM to 10 pM, from 750 aM to 1 pM, from 1 fM to 1 nM,
from 1 fM to 500 pM, from 1 fM to 200 pM, from 1 fM to 100 pM, from
1 fM to 10 pM, from 1 fM to 1 pM, from 500 fM to 500 pM, from 500
fM to 200 pM, from 500 fM to 100 pM, from 500 fM to 10 pM, from 500
fM to 1 pM, from 800 fM to 1 nM, from 800 fM to 500 pM, from 800 fM
to 200 pM, from 800 fM to 100 pM, from 800 fM to 10 pM, from 800 fM
to 1 pM, from 1 pM to 1 nM, from 1 pM to 500 pM, from 1 pM to 200
pM, from 1 pM to 100 pM, or from 1 pM to 10 pM) (where the
concentration refers to the threshold concentration of target DNA
at which the target DNA can be detected). In some cases, a method
of the present disclosure has a threshold of detection in a range
of from 1 aM to 800 aM. In some cases, a method of the present
disclosure has a threshold of detection in a range of from 50 aM to
1 pM. In some cases, a method of the present disclosure has a
threshold of detection in a range of from 50 aM to 500 fM.
[0418] In some cases, the minimum concentration at which a target
DNA can be detected in a sample is in a range of from 1 aM to 1 nM
(e.g., from 1 aM to 500 pM, from 1 aM to 200 pM, from 1 aM to 100
pM, from 1 aM to 10 pM, from 1 aM to 1 pM, from 100 aM to 1 nM,
from 100 aM to 500 pM, from 100 aM to 200 pM, from 100 aM to 100
pM, from 100 aM to 10 pM, from 100 aM to 1 pM, from 250 aM to 1 nM,
from 250 aM to 500 pM, from 250 aM to 200 pM, from 250 aM to 100
pM, from 250 aM to 10 pM, from 250 aM to 1 pM, from 500 aM to 1 nM,
from 500 aM to 500 pM, from 500 aM to 200 pM, from 500 aM to 100
pM, from 500 aM to 10 pM, from 500 aM to 1 pM, from 750 aM to 1 nM,
from 750 aM to 500 pM, from 750 aM to 200 pM, from 750 aM to 100
pM, from 750 aM to 10 pM, from 750 aM to 1 pM, from 1 fM to 1 nM,
from 1 fM to 500 pM, from 1 fM to 200 pM, from 1 fM to 100 pM, from
1 fM to 10 pM, from 1 fM to 1 pM, from 500 fM to 500 pM, from 500
fM to 200 pM, from 500 fM to 100 pM, from 500 fM to 10 pM, from 500
fM to 1 pM, from 800 fM to 1 nM, from 800 fM to 500 pM, from 800 fM
to 200 pM, from 800 fM to 100 pM, from 800 fM to 10 pM, from 800 fM
to 1 pM, from 1 pM to 1 nM, from 1 pM to 500 pM, from 1 pM to 200
pM, from 1 pM to 100 pM, or from 1 pM to 10 pM). In some cases, the
minimum concentration at which a target DNA can be detected in a
sample is in a range of from 1 aM to 500 pM. In some cases, the
minimum concentration at which a target DNA can be detected in a
sample is in a range of from 100 aM to 500 pM.
[0419] In some cases, a subject composition or method exhibits an
attomolar (aM) sensitivity of detection. In some cases, a subject
composition or method exhibits a femtomolar (fM) sensitivity of
detection. In some cases, a subject composition or method exhibits
a picomolar (pM) sensitivity of detection. In some cases, a subject
composition or method exhibits a nanomolar (nM) sensitivity of
detection.
Target DNA
[0420] A target DNA can be single stranded (ssDNA) or double
stranded (dsDNA). When the target DNA is single stranded, there is
no preference or requirement for a PAM sequence in the target DNA.
However, when the target DNA is dsDNA, a PAM is usually present
adjacent to the target sequence of the target DNA (e.g., see
discussion of the PAM elsewhere herein). The source of the target
DNA can be the same as the source of the sample, e.g., as described
below.
[0421] The source of the target DNA can be any source. In some
cases, the target DNA is a viral DNA (e.g., a genomic DNA of a DNA
virus). As such, subject method can be for detecting the presence
of a viral DNA amongst a population of nucleic acids (e.g., in a
sample). A subject method can also be used for the cleavage of
non-target ssDNAs in the present of a target DNA. For example, if a
method takes place in a cell, a subject method can be used to
promiscuously cleave non-target ssDNAs in the cell (ssDNAs that do
not hybridize with the guide sequence of the guide RNA) when a
particular target DNA is present in the cell (e.g., when the cell
is infected with a virus and viral target DNA is detected).
[0422] Examples of possible target DNAs include, but are not
limited to, viral DNAs such as: a papovavirus (e.g., human
papillomavirus (HPV), polyomavirus); a hepadnavirus (e.g.,
Hepatitis B Virus (HBV)); a herpesvirus (e.g., herpes simplex virus
(HSV), varicella zoster virus (VZV), epstein-barr virus (EBV),
cytomegalovirus (CMV), herpes lymphotropic virus, Pityriasis Rosea,
kaposi's sarcoma-associated herpesvirus); an adenovirus (e.g.,
atadenovirus, aviadenovirus, ichtadenovirus, mastadenovirus,
siadenovirus); a poxvirus (e.g., smallpox, vaccinia virus, cowpox
virus, monkeypox virus, orf virus, pseudocowpox, bovine papular
stomatitis virus; tanapox virus, yaba monkey tumor virus; molluscum
contagiosum virus (MCV)); a parvovirus (e.g., adeno-associated
virus (AAV), Parvovirus B19, human bocavirus, bufavirus, human
parv4 G1); Geminiviridae; Nanoviridae; Phycodnaviridae; and the
like. In some cases, the target DNA is parasite DNA. In some cases,
the target DNA is bacterial DNA, e.g., DNA of a pathogenic
bacterium.
Samples
[0423] A subject sample includes nucleic acid (e.g., a plurality of
nucleic acids). The term "plurality" is used herein to mean two or
more. Thus, in some cases, a sample includes two or more (e.g., 3
or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or
more, 500 or more, 1,000 or more, or 5,000 or more) nucleic acids
(e.g., DNAs). A subject method can be used as a very sensitive way
to detect a target DNA present in a sample (e.g., in a complex
mixture of nucleic acids such as DNAs). In some cases, the sample
includes 5 or more DNAs (e.g., 10 or more, 20 or more, 50 or more,
100 or more, 500 or more, 1,000 or more, or 5,000 or more DNAs)
that differ from one another in sequence. In some cases, the sample
includes 10 or more, 20 or more, 50 or more, 100 or more, 500 or
more, 10.sup.3 or more, 5.times.10.sup.3 or more, 10.sup.4 or more,
5.times.10.sup.4 or more, 10.sup.5 or more, 5.times.10.sup.5 or
more, 10.sup.6 or more 5.times.10.sup.6 or more, or 10' or more,
DNAs. In some cases, the sample comprises from 10 to 20, from 20 to
50, from 50 to 100, from 100 to 500, from 500 to 10.sup.3, from
10.sup.3 to 5.times.10.sup.3, from 5.times.10.sup.3 to 10.sup.4,
from 10.sup.4 to 5.times.10.sup.4, from 5.times.10.sup.4 to
10.sup.5, from 10.sup.5 to 5.times.10.sup.5, from 5.times.10.sup.5
to 10.sup.6, from 10.sup.6 to 5.times.10.sup.6, or from
5.times.10.sup.6 to 10', or more than 10', DNAs. In some cases, the
sample comprises from 5 to 10' DNAs (e.g., that differ from one
another in sequence)(e.g., from 5 to 10.sup.6, from 5 to 10.sup.5,
from 5 to 50,000, from 5 to 30,000, from 10 to 10.sup.6, from 10 to
10.sup.5, from 10 to 50,000, from 10 to 30,000, from 20 to
10.sup.6, from 20 to 10.sup.5, from 20 to 50,000, or from 20 to
30,000 DNAs). In some cases, the sample includes 20 or more DNAs
that differ from one another in sequence. In some cases, the sample
includes DNAs from a cell lysate (e.g., a eukaryotic cell lysate, a
mammalian cell lysate, a human cell lysate, a prokaryotic cell
lysate, a plant cell lysate, and the like). For example, in some
cases the sample includes DNA from a cell such as a eukaryotic
cell, e.g., a mammalian cell such as a human cell.
[0424] The term "sample" is used herein to mean any sample that
includes DNA (e.g., in order to determine whether a target DNA is
present among a population of DNAs). The sample can be derived from
any source, e.g., the sample can be a synthetic combination of
purified DNAs; the sample can be a cell lysate, an DNA-enriched
cell lysate, or DNAs isolated and/or purified from a cell lysate.
The sample can be from a patient (e.g., for the purpose of
diagnosis). The sample can be from permeabilized cells. The sample
can be from crosslinked cells. The sample can be in tissue
sections. The sample can be from tissues prepared by crosslinking
followed by delipidation and adjustment to make a uniform
refractive index. Examples of tissue preparation by crosslinking
followed by delipidation and adjustment to make a uniform
refractive index have been described in, for example, Shah et al.,
Development (2016) 143, 2862-2867 doi:10.1242/dev.138560.
[0425] A "sample" can include a target DNA and a plurality of
non-target DNAs. In some cases, the target DNA is present in the
sample at one copy per 10 non-target DNAs, one copy per 20
non-target DNAs, one copy per 25 non-target DNAs, one copy per 50
non-target DNAs, one copy per 100 non-target DNAs, one copy per 500
non-target DNAs, one copy per 10.sup.3 non-target DNAs, one copy
per 5.times.10.sup.3 non-target DNAs, one copy per 10.sup.4
non-target DNAs, one copy per 5.times.10.sup.4 non-target DNAs, one
copy per 10.sup.5 non-target DNAs, one copy per 5.times.10.sup.5
non-target DNAs, one copy per 10.sup.6 non-target DNAs, or less
than one copy per 10.sup.6 non-target DNAs. In some cases, the
target DNA is present in the sample at from one copy per 10
non-target DNAs to 1 copy per 20 non-target DNAs, from 1 copy per
20 non-target DNAs to 1 copy per 50 non-target DNAs, from 1 copy
per 50 non-target DNAs to 1 copy per 100 non-target DNAs, from 1
copy per 100 non-target DNAs to 1 copy per 500 non-target DNAs,
from 1 copy per 500 non-target DNAs to 1 copy per 10.sup.3
non-target DNAs, from 1 copy per 10.sup.3 non-target DNAs to 1 copy
per 5.times.10.sup.3 non-target DNAs, from 1 copy per
5.times.10.sup.3 non-target DNAs to 1 copy per 10.sup.4 non-target
DNAs, from 1 copy per 10.sup.4 non-target DNAs to 1 copy per
10.sup.5 non-target DNAs, from 1 copy per 10.sup.5 non-target DNAs
to 1 copy per 10.sup.6 non-target DNAs, or from 1 copy per 10.sup.6
non-target DNAs to 1 copy per 10.sup.7 non-target DNAs.
[0426] Suitable samples include but are not limited to saliva,
blood, serum, plasma, urine, aspirate, and biopsy samples. Thus,
the term "sample" with respect to a patient encompasses blood and
other liquid samples of biological origin, solid tissue samples
such as a biopsy specimen or tissue cultures or cells derived
therefrom and the progeny thereof. The definition also includes
samples that have been manipulated in any way after their
procurement, such as by treatment with reagents; washed; or
enrichment for certain cell populations, such as cancer cells. The
definition also includes sample that have been enriched for
particular types of molecules, e.g., DNAs. The term "sample"
encompasses biological samples such as a clinical sample such as
blood, plasma, serum, aspirate, cerebral spinal fluid (CSF), and
also includes tissue obtained by surgical resection, tissue
obtained by biopsy, cells in culture, cell supernatants, cell
lysates, tissue samples, organs, bone marrow, and the like. A
"biological sample" includes biological fluids derived therefrom
(e.g., cancerous cell, infected cell, etc.), e.g., a sample
comprising DNAs that is obtained from such cells (e.g., a cell
lysate or other cell extract comprising DNAs).
[0427] A sample can comprise, or can be obtained from, any of a
variety of cells, tissues, organs, or acellular fluids. Suitable
sample sources include eukaryotic cells, bacterial cells, and
archaeal cells. Suitable sample sources include single-celled
organisms and multi-cellular organisms. Suitable sample sources
include single-cell eukaryotic organisms; a plant or a plant cell;
an algal cell, e.g., Botryococcus braunii, Chlamydomonas
reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa,
Sargassum patens, C. agardh, and the like; a fungal cell (e.g., a
yeast cell); an animal cell, tissue, or organ; a cell, tissue, or
organ from an invertebrate animal (e.g. fruit fly, cnidarian,
echinoderm, nematode, an insect, an arachnid, etc.); a cell,
tissue, fluid, or organ from a vertebrate animal (e.g., fish,
amphibian, reptile, bird, mammal); a cell, tissue, fluid, or organ
from a mammal (e.g., a human; a non-human primate; an ungulate; a
feline; a bovine; an ovine; a caprine; etc.). Suitable sample
sources include nematodes, protozoans, and the like. Suitable
sample sources include parasites such as helminths, malarial
parasites, etc.
[0428] Suitable sample sources include a cell, tissue, or organism
of any of the six kingdoms, e.g., Bacteria (e.g., Eubacteria);
Archaebacteria; Protista; Fungi; Plantae; and Animalia. Suitable
sample sources include plant-like members of the kingdom Protista,
including, but not limited to, algae (e.g., green algae, red algae,
glaucophytes, cyanobacteria); fungus-like members of Protista,
e.g., slime molds, water molds, etc.; animal-like members of
Protista, e.g., flagellates (e.g., Euglena), amoeboids (e.g.,
amoeba), sporozoans (e.g, Apicomplexa, Myxozoa, Microsporidia), and
ciliates (e.g., Paramecium). Suitable sample sources include
include members of the kingdom Fungi, including, but not limited
to, members of any of the phyla: Basidiomycota (club fungi; e.g.,
members of Agaricus, Amanita, Boletus, Cantherellus, etc.);
Ascomycota (sac fungi, including, e.g., Saccharomyces);
Mycophycophyta (lichens); Zygomycota (conjugation fungi); and
Deuteromycota. Suitable sample sources include include members of
the kingdom Plantae, including, but not limited to, members of any
of the following divisions: Bryophyta (e.g., mosses),
Anthocerotophyta (e.g., hornworts), Hepaticophyta (e.g.,
liverworts), Lycophyta (e.g., club mosses), Sphenophyta (e.g.,
horsetails), Psilophyta (e.g., whisk ferns), Ophioglossophyta,
Pterophyta (e.g., ferns), Cycadophyta, Gingkophyta, Pinophyta,
Gnetophyta, and Magnoliophyta (e.g., flowering plants). Suitable
sample sources include include members of the kingdom Animalia,
including, but not limited to, members of any of the following
phyla: Porifera (sponges); Placozoa; Orthonectida (parasites of
marine invertebrates); Rhombozoa; Cnidaria (corals, anemones,
jellyfish, sea pens, sea pansies, sea wasps); Ctenophora (comb
jellies); Platyhelminthes (flatworms); Nemertina (ribbon worms);
Ngathostomulida (jawed worms)p Gastrotricha; Rotifera; Priapulida;
Kinorhyncha; Loricifera; Acanthocephala; Entoprocta; Nemotoda;
Nematomorpha; Cycliophora; Mollusca (mollusks); Sipuncula (peanut
worms); Annelida (segmented worms); Tardigrada (water bears);
Onychophora (velvet worms); Arthropoda (including the subphyla:
Chelicerata, Myriapoda, Hexapoda, and Crustacea, where the
Chelicerata include, e.g., arachnids, Merostomata, and Pycnogonida,
where the Myriapoda include, e.g., Chilopoda (centipedes),
Diplopoda (millipedes), Paropoda, and Symphyla, where the Hexapoda
include insects, and where the Crustacea include shrimp, hill,
barnacles, etc.; Phoronida; Ectoprocta (moss animals); Brachiopoda;
Echinodermata (e.g. starfish, sea daisies, feather stars, sea
urchins, sea cucumbers, brittle stars, brittle baskets, etc.);
Chaetognatha (arrow worms); Hemichordata (acorn worms); and
Chordata. Suitable members of Chordata include any member of the
following subphyla: Urochordata (sea squirts; including Ascidiacea,
Thaliacea, and Larvacea); Cephalochordata (lancelets); Myxini
(hagfish); and Vertebrata, where members of Vertebrata include,
e.g., members of Petromyzontida (lampreys), Chondrichthyces
(cartilaginous fish), Actinopterygii (ray-finned fish), Actinista
(coelocanths), Dipnoi (lungfish), Reptilia (reptiles, e.g., snakes,
alligators, crocodiles, lizards, etc.), Ayes (birds); and Mammalian
(mammals) Suitable plants include any monocotyledon and any
dicotyledon.
[0429] Suitable sources of a sample include cells, fluid, tissue,
or organ taken from an organism; from a particular cell or group of
cells isolated from an organism; etc. For example, where the
organism is a plant, suitable sources include xylem, the phloem,
the cambium layer, leaves, roots, etc. Where the organism is an
animal, suitable sources include particular tissues (e.g., lung,
liver, heart, kidney, brain, spleen, skin, fetal tissue, etc.), or
a particular cell type (e.g., neuronal cells, epithelial cells,
endothelial cells, astrocytes, macrophages, glial cells, islet
cells, T lymphocytes, B lymphocytes, etc.).
[0430] In some cases, the source of the sample is a (or is
suspected of being a diseased cell, fluid, tissue, or organ. In
some cases, the source of the sample is a normal (non-diseased)
cell, fluid, tissue, or organ. In some cases, the source of the
sample is a (or is suspected of being) a pathogen-infected cell,
tissue, or organ. For example, the source of a sample can be an
individual who may or may not be infected--and the sample could be
any biological sample (e.g., blood, saliva, biopsy, plasma, serum,
bronchoalveolar lavage, sputum, a fecal sample, cerebrospinal
fluid, a fine needle aspirate, a swab sample (e.g., a buccal swab,
a cervical swab, a nasal swab), interstitial fluid, synovial fluid,
nasal discharge, tears, buffy coat, a mucous membrane sample, an
epithelial cell sample (e.g., epithelial cell scraping), etc.)
collected from the individual. In some cases, the sample is a
cell-free liquid sample. In some cases, the sample is a liquid
sample that can comprise cells. Pathogens include viruses, fungi,
helminths, protozoa, malarial parasites, Plasmodium parasites,
Toxoplasma parasites, Schistosoma parasites, and the like.
"Helminths" include roundworms, heartworms, and phytophagous
nematodes (Nematoda), flukes (Tematoda), Acanthocephala, and
tapeworms (Cestoda). Protozoan infections include infections from
Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic
dysentery, babesiosis, balantidial dysentery, Chaga's disease,
coccidiosis, malaria and toxoplasmosis. Examples of pathogens such
as parasitic/protozoan pathogens include, but are not limited to:
Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruzi and
Toxoplasma gondii. Fungal pathogens include, but are not limited
to: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides
immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and
Candida albicans. Pathogenic viruses include, e.g., human
immunodeficiency virus (e.g., HIV); influenza virus; dengue; West
Nile virus; herpes virus; yellow fever virus; Hepatitis C Virus;
Hepatitis A Virus; Hepatitis B Virus; papillomavirus; and the like.
Pathogenic viruses can include DNA viruses such as: a papovavirus
(e.g., human papillomavirus (HPV), polyomavirus); a hepadnavirus
(e.g., Hepatitis B Virus (HBV)); a herpesvirus (e.g., herpes
simplex virus (HSV), varicella zoster virus (VZV), Epstein-Barr
virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus,
Pityriasis Rosea, Kaposi's sarcoma-associated herpesvirus); an
adenovirus (e.g., atadenovirus, aviadenovirus, ichtadenovirus,
mastadenovirus, siadenovirus); a poxvirus (e.g., smallpox, vaccinia
virus, cowpox virus, monkeypox virus, orf virus, pseudocowpox,
bovine papular stomatitis virus; tanapox virus, yaba monkey tumor
virus; molluscum contagiosum virus (MCV)); a parvovirus (e.g.,
adeno-associated virus (AAV), Parvovirus B19, human bocavirus,
bufavirus, human parv4 G1); Geminiviridae; Nanoviridae;
Phycodnaviridae; and the like. Pathogens can include, e.g.,
DNAviruses (e.g.: a papovavirus (e.g., human papillomavirus (HPV),
polyomavirus); a hepadnavirus (e.g., Hepatitis B Virus (HBV)); a
herpesvirus (e.g., herpes simplex virus (HSV), varicella zoster
virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV),
herpes lymphotropic virus, Pityriasis Rosea, Kaposi's
sarcoma-associated herpesvirus); an adenovirus (e.g., atadenovirus,
aviadenovirus, ichtadenovirus, mastadenovirus, siadenovirus); a
poxvirus (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox
virus, orf virus, pseudocowpox, bovine papular stomatitis virus;
tanapox virus, yaba monkey tumor virus; molluscum contagiosum virus
(MCV)); a parvovirus (e.g., adeno-associated virus (AAV),
Parvovirus B19, human bocavirus, bufavirus, human parv4 G1);
Geminiviridae; Nanoviridae; Phycodnaviridae; and the like],
Mycobacterium tuberculosis, Streptococcus agalactiae,
methicillin-resistant Staphylococcus aureus, Legionella
pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria
gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus
neoformans, Histoplasma capsulatum, Hemophilus influenzae B,
Treponema pallidum, Lyme disease spirochetes, Pseudomonas
aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus,
influenza virus, cytomegalovirus, herpes simplex virus I, herpes
simplex virus II, human serum parvo-like virus, respiratory
syncytial virus, varicella-zoster virus, hepatitis B virus,
hepatitis C virus, measles virus, adenovirus, human T-cell leukemia
viruses, Epstein-Barr virus, murine leukemia virus, mumps virus,
vesicular stomatitis virus, Sindbis virus, lymphocytic
choriomeningitis virus, wart virus, blue tongue virus, Sendai
virus, feline leukemia virus, Reovirus, polio virus, simian virus
40, mouse mammary tumor virus, dengue virus, rubella virus, West
Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma
gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma
rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma
japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus,
Leishmania tropica, Mycobacterium tuberculosis, Trichinella
spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia
saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma
arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma
laidlawii, M. salivarium and M. pneumoniae.
Measuring a Detectable Signal
[0431] In some cases, a subject method includes a step of measuring
(e.g., measuring a detectable signal produced by Cas12J-mediated
ssDNA cleavage). Because a Cas12J polypeptide of the present
disclosure cleaves non-targeted ssDNA once activated, which occurs
when a guide RNA hybridizes with a target DNA in the presence of a
Cas12J effector protein, a detectable signal can be any signal that
is produced when ssDNA is cleaved. For example, in some cases, the
step of measuring can include one or more of: gold nanoparticle
based detection (e.g., see Xu et al., Angew Chem Int Ed Engl. 2007;
46(19):3468-70; and Xia et al., Proc Natl Acad Sci USA. 2010 Jun.
15; 107(24):10837-41), fluorescence polarization, colloid phase
transition/dispersion (e.g., Baksh et al., Nature. 2004 Jan. 8;
427(6970):139-41), electrochemical detection, semiconductor-based
sensing (e.g., Rothberg et al., Nature. 2011 Jul. 20;
475(7356):348-52; e.g., one could use a phosphatase to generate a
pH change after ssDNA cleavage reactions, by opening 2'-3' cyclic
phosphates, and by releasing inorganic phosphate into solution),
and detection of a labeled detector ssDNA (see elsewhere herein for
more details). The readout of such detection methods can be any
convenient readout. Examples of possible readouts include but are
not limited to: a measured amount of detectable fluorescent signal;
a visual analysis of bands on a gel (e.g., bands that represent
cleaved product versus uncleaved substrate), a visual or sensor
based detection of the presence or absence of a color (i.e., color
detection method), and the presence or absence of (or a particular
amount of) an electrical signal.
[0432] The measuring can in some cases be quantitative, e.g., in
the sense that the amount of signal detected can be used to
determine the amount of target DNA present in the sample. The
measuring can in some cases be qualitative, e.g., in the sense that
the presence or absence of detectable signal can indicate the
presence or absence of targeted DNA (e.g., virus, SNP, etc.). In
some cases, a detectable signal will not be present (e.g., above a
given threshold level) unless the targeted DNA(s) (e.g., virus,
SNP, etc.) is present above a particular threshold concentration.
In some cases, the threshold of detection can be titrated by
modifying the amount of Cas12J effector, guide RNA, sample volume,
and/or detector ssDNA (if one is used). As such, for example, as
would be understood by one of ordinary skill in the art, a number
of controls can be used if desired in order to set up one or more
reactions, each set up to detect a different threshold level of
target DNA, and thus such a series of reactions could be used to
determine the amount of target DNA present in a sample (e.g., one
could use such a series of reactions to determine that a target DNA
is present in the sample `at a concentration of at least X`).
[0433] Examples of uses of a detection method of the present
disclosure include, e.g., single nucleotide polymorphism (SNP)
detection, cancer screening, detection of bacterial infection,
detection of antibiotic resistance, detection of viral infection,
and the like. The compositions and methods of this disclosure can
be used to detect any DNA target. For example, any virus that
integrates nucleic acid material into the genome can be detected
because a subject sample can include cellular genomic DNA--and the
guide RNA can be designed to detect integrated nucleotide
sequence.
[0434] In some cases, a method of the present disclosure can be
used to determine the amount of a target DNA in a sample (e.g., a
sample comprising the target DNA and a plurality of non-target
DNAs). Determining the amount of a target DNA in a sample can
comprise comparing the amount of detectable signal generated from a
test sample to the amount of detectable signal generated from a
reference sample. Determining the amount of a target DNA in a
sample can comprise: measuring the detectable signal to generate a
test measurement; measuring a detectable signal produced by a
reference sample to generate a reference measurement; and comparing
the test measurement to the reference measurement to determine an
amount of target DNA present in the sample.
[0435] For example, in some cases, a method of the present
disclosure for determining the amount of a target DNA in a sample
comprises: a) contacting the sample (e.g., a sample comprising the
target DNA and a plurality of non-target DNAs) with: (i) a guide
RNA that hybridizes with the target DNA, (ii) a Cas12J polypeptide
of the present disclosure that cleaves RNAs present in the sample,
and (iii) a detector ssDNA; b) measuring a detectable signal
produced by Cas12J-mediated ssDNA cleavage (e.g., cleavage of the
detector ssDNA), generating a test measurement; c) measuring a
detectable signal produced by a reference sample to generate a
reference measurement; and d) comparing the test measurement to the
reference measurement to determine an amount of target DNA present
in the sample.
[0436] As another example, in some cases, a method of the present
disclosure for determining the amount of a target DNA in a sample
comprises: a) contacting the sample (e.g., a sample comprising the
target DNA and a plurality of non-target DNAs) with: i) a precursor
guide RNA array comprising two or more guide RNAs each of which has
a different guide sequence; (ii) a Cas12J polypeptide of the
present disclosure that cleaves the precursor guide RNA array into
individual guide RNAs, and also cleaves RNAs of the sample; and
(iii) a detector ssDNA; b) measuring a detectable signal produced
by Cas12J-mediated ssDNA cleavage (e.g., cleavage of the detector
ssDNA), generating a test measurement; c) measuring a detectable
signal produced by each of two or more reference samples to
generate two or more reference measurements; and d) comparing the
test measurement to the reference measurements to determine an
amount of target DNA present in the sample.
Amplification of Nucleic Acids in the Sample
[0437] In some embodiments, sensitivity of a subject composition
and/or method (e.g., for detecting the presence of a target DNA,
such as viral DNA or a SNP, in cellular genomic DNA) can be
increased by coupling detection with nucleic acid amplification. In
some cases, the nucleic acids in a sample are amplified prior to
contact with a Cas12J polypeptide of the present disclosure that
cleaved ssDNA (e.g., amplification of nucleic acids in the sample
can begin prior to contact with a Cas12J polypeptide of the present
disclosure). In some cases, the nucleic acids in a sample are
amplified simultaneously with contact with a Cas12J polypeptide of
the present disclosure. For example, in some cases, a subject
method includes amplifying nucleic acids of a sample (e.g., by
contacting the sample with amplification components) prior to
contacting the amplified sample with a Cas12J polypeptide of the
present disclosure. In some cases, a subject method includes
contacting a sample with amplification components at the same time
(simultaneous with) that the sample is contacted with a Cas12J
polypeptide of the present disclosure. If all components are added
simultaneously (amplification components and detection components
such as a Cas12J polypeptide of the present disclosure, a guide
RNA, and a detector DNA), it is possible that the trans-cleavage
activity of the Cas12J will begin to degrade the nucleic acids of
the sample at the same time the nucleic acids are undergoing
amplification. However, even if this is the case, amplifying and
detecting simultaneously can still increase sensitivity compared to
performing the method without amplification.
[0438] In some cases, specific sequences (e.g., sequences of a
virus, sequences that include a SNP of interest) are amplified from
the sample, e.g., using primers. As such, a sequence to which the
guide RNA will hybridize can be amplified in order to increase
sensitivity of a subject detection method--this could achieve
biased amplification of a desired sequence in order to increase the
number of copies of the sequence of interest present in the sample
relative to other sequences present in the sample. As one
illustrative example, if a subject method is being used to
determine whether a given sample includes a particular virus (or a
particular SNP), a desired region of viral sequence (or non-viral
genomic sequence) can be amplified, and the region amplified will
include the sequence that would hybridize to the guide RNA if the
viral sequence (or SNP) were in fact present in the sample.
[0439] As noted, in some cases the nucleic acids are amplified
(e.g., by contact with amplification components) prior to
contacting the amplified nucleic acids with a Cas12J polypeptide of
the present disclosure. In some cases, amplification occurs for 10
seconds or more, (e.g., 30 seconds or more, 45 seconds or more, 1
minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or
more, 5 minutes or more, 7.5 minutes or more, 10 minutes or more,
etc.) prior to contact with a Cas12J polypeptide of the present
disclosure. In some cases, amplification occurs for 2 minutes or
more (e.g., 3 minutes or more, 4 minutes or more, 5 minutes or
more, 7.5 minutes or more, 10 minutes or more, etc.) prior to
contact with a Cas12J polypeptide of the present disclosure. In
some cases, amplification occurs for a period of time in a range of
from 10 seconds to 60 minutes (e.g., 10 seconds to 40 minutes, 10
seconds to 30 minutes, 10 seconds to 20 minutes, 10 seconds to 15
minutes, 10 seconds to 10 minutes, 10 seconds to 5 minutes, 30
seconds to 40 minutes, 30 seconds to 30 minutes, 30 seconds to 20
minutes, 30 seconds to 15 minutes, 30 seconds to 10 minutes, 30
seconds to 5 minutes, 1 minute to 40 minutes, 1 minute to 30
minutes, 1 minute to 20 minutes, 1 minute to 15 minutes, 1 minute
to 10 minutes, 1 minute to 5 minutes, 2 minutes to 40 minutes, 2
minutes to 30 minutes, 2 minutes to 20 minutes, 2 minutes to 15
minutes, 2 minutes to 10 minutes, 2 minutes to 5 minutes, 5 minutes
to 40 minutes, 5 minutes to 30 minutes, 5 minutes to 20 minutes, 5
minutes to 15 minutes, or 5 minutes to 10 minutes). In some cases,
amplification occurs for a period of time in a range of from 5
minutes to 15 minutes. In some cases, amplification occurs for a
period of time in a range of from 7 minutes to 12 minutes.
[0440] In some cases, a sample is contacted with amplification
components at the same time as contact with a Cas12J polypeptide of
the present disclosure. In some such cases, the Cas12J protein is
inactive at the time of contact and is activated once nucleic acids
in the sample have been amplified.
[0441] Various amplification methods and components will be known
to one of ordinary skill in the art and any convenient method can
be used (see, e.g., Zanoli and Spoto, Biosensors (Basel). 2013
March; 3(1): 18-43; Gill and Ghaemi, Nucleosides, Nucleotides, and
Nucleic Acids, 2008, 27: 224-243; Craw and Balachandrana, Lab Chip,
2012, 12, 2469-2486; which are herein incorporated by reference in
their entirety). Nucleic acid amplification can comprise polymerase
chain reaction (PCR), reverse transcription PCR (RT-PCR),
quantitative PCR (qPCR), reverse transcription qPCR (RT-qPCR),
nested PCR, multiplex PCR, asymmetric PCR, touchdown PCR, random
primer PCR, hemi-nested PCR, polymerase cycling assembly (PCA),
colony PCR, ligase chain reaction (LCR), digital PCR, methylation
specific-PCR (MSP), co-amplification at lower denaturation
temperature-PCR (COLD-PCR), allele-specific PCR,
intersequence-specific PCR (ISS-PCR), whole genome amplification
(WGA), inverse PCR, and thermal asymmetric interlaced PCR
(TAIL-PCR).
[0442] In some cases, the amplification is isothermal
amplification. The term "isothermal amplification" indicates a
method of nucleic acid (e.g., DNA) amplification (e.g., using
enzymatic chain reaction) that can use a single temperature
incubation thereby obviating the need for a thermal cycler.
Isothermal amplification is a form of nucleic acid amplification
which does not rely on the thermal denaturation of the target
nucleic acid during the amplification reaction and hence may not
require multiple rapid changes in temperature. Isothermal nucleic
acid amplification methods can therefore be carried out inside or
outside of a laboratory environment. By combining with a reverse
transcription step, these amplification methods can be used to
isothermally amplify RNA.
[0443] Examples of isothermal amplification methods include but are
not limited to: loop-mediated isothermal Amplification (LAMP),
helicase-dependent Amplification (HDA), recombinase polymerase
amplification (RPA), strand displacement amplification (SDA),
nucleic acid sequence-based amplification (NASBA), transcription
mediated amplification (TMA), nicking enzyme amplification reaction
(NEAR), rolling circle amplification (RCA), multiple displacement
amplification (MDA), Ramification (RAM), circular
helicase-dependent amplification (cHDA), single primer isothermal
amplification (SPIA), signal mediated amplification of RNA
technology (SMART), self-sustained sequence replication (3SR),
genome exponential amplification reaction (GEAR) and isothermal
multiple displacement amplification (IMDA).
[0444] In some cases, the amplification is recombinase polymerase
amplification (RPA) (see, e.g., U.S. Pat. Nos. 8,030,000;
8,426,134; 8,945,845; 9,309,502; and 9,663,820, which are hereby
incorporated by reference in their entirety). Recombinase
polymerase amplification (RPA) uses two opposing primers (much like
PCR) and employs three enzymes--a recombinase, a single-stranded
DNA-binding protein (SSB) and a strand-displacing polymerase. The
recombinase pairs oligonucleotide primers with homologous sequence
in duplex DNA, SSB binds to displaced strands of DNA to prevent the
primers from being displaced, and the strand displacing polymerase
begins DNA synthesis where the primer has bound to the target DNA.
Adding a reverse transcriptase enzyme to an RPA reaction can
facilitate detection RNA as well as DNA, without the need for a
separate step to produce cDNA. One example of components for an RPA
reaction is as follows (see, e.g., U.S. Pat. Nos. 8,030,000;
8,426,134; 8,945,845; 9,309,502; 9,663,820): 50 mM Tris pH 8.4, 80
mM Potassium actetate, 10 mM Magnesium acetate, 2 mM dithiothreitol
(DTT), 5% PEG compound (Carbowax-20M), 3 mM ATP, 30 mM
Phosphocreatine, 100 ng/.mu.1 creatine kinase, 420 ng/.mu.1 gp32,
140 ng/.mu.l UvsX, 35 ng/.mu.l UvsY, 2000M dNTPs, 300 nM each
oligonucleotide, 35 ng/.mu.l Bsu polymerase, and a nucleic
acid-containing sample).
[0445] In a transcription mediated amplification (TMA), an RNA
polymerase is used to make RNA from a promoter engineered in the
primer region, and then a reverse transcriptase synthesizes cDNA
from the primer. A third enzyme, e.g., Rnase H can then be used to
degrade the RNA target from cDNA without the heat-denatured step.
This amplification technique is similar to Self-Sustained Sequence
Replication (3SR) and Nucleic Acid Sequence Based Amplification
(NASBA), but varies in the enzymes employed. For another example,
helicase-dependent amplification (HDA) utilizes a thermostable
helicase (Tte-UvrD) rather than heat to unwind dsDNA to create
single-strands that are then available for hybridization and
extension of primers by polymerase. For yet another example, a loop
mediated amplification (LAMP) employs a thermostable polymerase
with strand displacement capabilities and a set of four or more
specific designed primers. Each primer is designed to have hairpin
ends that, once displaced, snap into a hairpin to facilitate
self-priming and further polymerase extension. In a LAMP reaction,
though the reaction proceeds under isothermal conditions, an
initial heat denaturation step is required for double-stranded
targets. In addition, amplification yields a ladder pattern of
various length products. For yet another example, a strand
displacement amplification (SDA) combines the ability of a
restriction endonuclease to nick the unmodified strand of its
target DNA and an exonuclease-deficient DNA polymerase to extend
the 3' end at the nick and displace the downstream DNA strand.
Detector DNA
[0446] In some cases, a subject method includes contacting a sample
(e.g., a sample comprising a target DNA and a plurality of
non-target ssDNAs) with: i) a Cas12J polypeptide of the present
disclosure; ii) a guide RNA (or precursor guide RNA array); and
iii) a detector DNA that is single stranded and does not hybridize
with the guide sequence of the guide RNA. For example, in some
cases, a subject method includes contacting a sample with a labeled
single stranded detector DNA (detector ssDNA) that includes a
fluorescence-emitting dye pair; the Cas12J polypeptide cleaves the
labeled detector ssDNA after it is activated (by binding to the
guide RNA in the context of the guide RNA hybridizing to a target
DNA); and the detectable signal that is measured is produced by the
fluorescence-emitting dye pair. For example, in some cases, a
subject method includes contacting a sample with a labeled detector
ssDNA comprising a fluorescence resonance energy transfer (FRET)
pair or a quencher/fluor pair, or both. In some cases, a subject
method includes contacting a sample with a labeled detector ssDNA
comprising a FRET pair. In some cases, a subject method includes
contacting a sample with a labeled detector ssDNA comprising a
fluor/quencher pair.
[0447] Fluorescence-emitting dye pairs comprise a FRET pair or a
quencher/fluor pair. In both cases of a FRET pair and a
quencher/fluor pair, the emission spectrum of one of the dyes
overlaps a region of the absorption spectrum of the other dye in
the pair. As used herein, the term "fluorescence-emitting dye pair"
is a generic term used to encompass both a "fluorescence resonance
energy transfer (FRET) pair" and a "quencher/fluor pair," both of
which terms are discussed in more detail below. The term
"fluorescence-emitting dye pair" is used interchangeably with the
phrase "a FRET pair and/or a quencher/fluor pair."
[0448] In some cases (e.g., when the detector ssDNA includes a FRET
pair) the labeled detector ssDNA produces an amount of detectable
signal prior to being cleaved, and the amount of detectable signal
that is measured is reduced when the labeled detector ssDNA is
cleaved. In some cases, the labeled detector ssDNA produces a first
detectable signal prior to being cleaved (e.g., from a FRET pair)
and a second detectable signal when the labeled detector ssDNA is
cleaved (e.g., from a quencher/fluor pair). As such, in some cases,
the labeled detector ssDNA comprises a FRET pair and a
quencher/fluor pair.
[0449] In some cases, the labeled detector ssDNA comprises a FRET
pair. FRET is a process by which radiationless transfer of energy
occurs from an excited state fluorophore to a second chromophore in
close proximity. The range over which the energy transfer can take
place is limited to approximately 10 nanometers (100 angstroms),
and the efficiency of transfer is extremely sensitive to the
separation distance between fluorophores. Thus, as used herein, the
term "FRET" ("fluorescence resonance energy transfer"; also known
as "Forster resonance energy transfer") refers to a physical
phenomenon involving a donor fluorophore and a matching acceptor
fluorophore selected so that the emission spectrum of the donor
overlaps the excitation spectrum of the acceptor, and further
selected so that when donor and acceptor are in close proximity
(usually 10 nm or less) to one another, excitation of the donor
will cause excitation of and emission from the acceptor, as some of
the energy passes from donor to acceptor via a quantum coupling
effect. Thus, a FRET signal serves as a proximity gauge of the
donor and acceptor; only when they are in close proximity to one
another is a signal generated. The FRET donor moiety (e.g., donor
fluorophore) and FRET acceptor moiety (e.g., acceptor fluorophore)
are collectively referred to herein as a "FRET pair".
[0450] The donor-acceptor pair (a FRET donor moiety and a FRET
acceptor moiety) is referred to herein as a "FRET pair" or a
"signal FRET pair." Thus, in some cases, a subject labeled detector
ssDNA includes two signal partners (a signal pair), when one signal
partner is a FRET donor moiety and the other signal partner is a
FRET acceptor moiety. A subject labeled detector ssDNA that
includes such a FRET pair (a FRET donor moiety and a FRET acceptor
moiety) will thus exhibit a detectable signal (a FRET signal) when
the signal partners are in close proximity (e.g., while on the same
RNA molecule), but the signal will be reduced (or absent) when the
partners are separated (e.g., after cleavage of the RNA molecule by
a Cas12J polypeptide of the present disclosure).
[0451] FRET donor and acceptor moieties (FRET pairs) will be known
to one of ordinary skill in the art and any convenient FRET pair
(e.g., any convenient donor and acceptor moiety pair) can be used.
Examples of suitable FRET pairs include but are not limited to
those presented in Table 1. See also: Bajar et al. Sensors (Basel).
2016 Sep. 14; 16(9); and Abraham et al. PLoS One. 2015 Aug. 3;
10(8):e0134436.
TABLE-US-00014 TABLE 1 Examples of FRET pairs (donor and acceptor
FRET moieties) Donor Acceptor Tryptophan Dansyl IAEDANS (1) DDPM
(2) BFP DsRFP Dansyl Fluorescein isothiocyanate (FITC) Dansyl
Octadecylrhodamine Cyan fluorescent protein (CFP) Green fluorescent
protein (GFP) CF (3) Texas Red Fluorescein Tetramethylrhodamine Cy3
Cy5 GFP Yellow fluorescent protein (YFP) BODIPY FL (4) BODIPY FL
(4) Rhodamine 110 Cy3 Rhodamine 6G Malachite Green FITC Eosin
Thiosemicarbazide B-Phycoerythrin Cy5 Cy5 Cy5.5 (1)
5-(2-iodoacetylaminoethyl)aminonaphthalene-1-sulfonic acid (2)
N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (3)
carboxyfluorescein succinimidyl ester (4)
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
[0452] In some cases, a detectable signal is produced when the
labeled detector ssDNA is cleaved (e.g., in some cases, the labeled
detector ssDNA comprises a quencher/fluor pair). One signal partner
of a signal quenching pair produces a detectable signal and the
other signal partner is a quencher moiety that quenches the
detectable signal of the first signal partner (i.e., the quencher
moiety quenches the signal of the signal moiety such that the
signal from the signal moiety is reduced (quenched) when the signal
partners are in proximity to one another, e.g., when the signal
partners of the signal pair are in close proximity).
[0453] For example, in some cases, an amount of detectable signal
increases when the labeled detector ssDNA is cleaved. For example,
in some cases, the signal exhibited by one signal partner (a signal
moiety) is quenched by the other signal partner (a quencher signal
moiety), e.g., when both are present on the same ssDNA molecule
prior to cleavage by a Cas12J polypeptide of the present
disclosure). Such a signal pair is referred to herein as a
"quencher/fluor pair", "quenching pair", or "signal quenching
pair." For example, in some cases, one signal partner (e.g., the
first signal partner) is a signal moiety that produces a detectable
signal that is quenched by the second signal partner (e.g., a
quencher moiety). The signal partners of such a quencher/fluor pair
will thus produce a detectable signal when the partners are
separated (e.g., after cleavage of the detector ssDNA by a Cas12J
polypeptide of the present disclosure), but the signal will be
quenched when the partners are in close proximity (e.g., prior to
cleavage of the detector ssDNA by a Cas12J polypeptide of the
present disclosure).
[0454] A quencher moiety can quench a signal from the signal moiety
(e.g., prior to cleave of the detector ssDNA by a Cas12J
polypeptide of the present disclosure) to various degrees. In some
cases, a quencher moiety quenches the signal from the signal moiety
where the signal detected in the presence of the quencher moiety
(when the signal partners are in proximity to one another) is 95%
or less of the signal detected in the absence of the quencher
moiety (when the signal partners are separated). For example, in
some cases, the signal detected in the presence of the quencher
moiety can be 90% or less, 80% or less, 70% or less, 60% or less,
50% or less, 40% or less, 30% or less, 20% or less, 15% or less,
10% or less, or 5% or less of the signal detected in the absence of
the quencher moiety. In some cases, no signal (e.g., above
background) is detected in the presence of the quencher moiety.
[0455] In some cases, the signal detected in the absence of the
quencher moiety (when the signal partners are separated) is at
least 1.2 fold greater (e.g., at least 1.3 fold, at least 1.5 fold,
at least 1.7 fold, at least 2 fold, at least 2.5 fold, at least 3
fold, at least 3.5 fold, at least 4 fold, at least 5 fold, at least
7 fold, at least 10 fold, at least 20 fold, or at least 50 fold
greater) than the signal detected in the presence of the quencher
moiety (when the signal partners are in proximity to one
another).
[0456] In some cases, the signal moiety is a fluorescent label. In
some such cases, the quencher moiety quenches the signal (the light
signal) from the fluorescent label (e.g., by absorbing energy in
the emission spectra of the label). Thus, when the quencher moiety
is not in proximity with the signal moiety, the emission (the
signal) from the fluorescent label is detectable because the signal
is not absorbed by the quencher moiety. Any convenient donor
acceptor pair (signal moiety/quencher moiety pair) can be used and
many suitable pairs are known in the art.
[0457] In some cases, the quencher moiety absorbs energy from the
signal moiety (also referred to herein as a "detectable label") and
then emits a signal (e.g., light at a different wavelength). Thus,
in some cases, the quencher moiety is itself a signal moiety (e.g.,
a signal moiety can be 6-carboxyfluorescein while the quencher
moiety can be 6-carboxy-tetramethylrhodamine), and in some such
cases, the pair could also be a FRET pair. In some cases, a
quencher moiety is a dark quencher. A dark quencher can absorb
excitation energy and dissipate the energy in a different way
(e.g., as heat). Thus, a dark quencher has minimal to no
fluorescence of its own (does not emit fluorescence). Examples of
dark quenchers are further described in U.S. Pat. Nos. 8,822,673
and 8,586,718; U.S. patent publications 20140378330, 20140349295,
and 20140194611; and international patent applications: WO200142505
and WO200186001, all if which are hereby incorporated by reference
in their entirety.
[0458] Examples of fluorescent labels include, but are not limited
to: an Alexa Fluor.RTM. dye, an ATTO dye (e.g., ATTO 390, ATTO 425,
ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO
Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO
Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13,
ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO
655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740),
a DyLight dye, a cyanine dye (e.g., Cy2, Cy3, Cy3.5, Cy3b, Cy5,
Cy5.5, Cy7, Cy7.5), a FluoProbes dye, a Sulfo Cy dye, a Seta dye,
an IRIS Dye, a SeTau dye, an SRfluor dye, a Square dye, fluorescein
isothiocyanate (FITC), tetramethylrhodamine (TRITC), Texas Red,
Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, quantum
dots, and a tethered fluorescent protein.
[0459] In some cases, a detectable label is a fluorescent label
selected from: an Alexa Fluor.RTM. dye, an ATTO dye (e.g., ATTO
390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520,
ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B,
ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO
594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO
647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700,
ATTO 725, ATTO 740), a DyLight dye, a cyanine dye (e.g., Cy2, Cy3,
Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5), a FluoProbes dye, a Sulfo Cy
dye, a Seta dye, an IRIS Dye, a SeTau dye, an SRfluor dye, a Square
dye, fluorescein (FITC), tetramethylrhodamine (TRITC), Texas Red,
Oregon Green, Pacific Blue, Pacific Green, and Pacific Orange.
[0460] In some cases, a detectable label is a fluorescent label
selected from: an Alexa Fluor.RTM. dye, an ATTO dye (e.g., ATTO
390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520,
ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B,
ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO
594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO
647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700,
ATTO 725, ATTO 740), a DyLight dye, a cyanine dye (e.g., Cy2, Cy3,
Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5), a FluoProbes dye, a Sulfo Cy
dye, a Seta dye, an IRIS Dye, a SeTau dye, an SRfluor dye, a Square
dye, fluorescein (FITC), tetramethylrhodamine (TRITC), Texas Red,
Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, a
quantum dot, and a tethered fluorescent protein.
[0461] Examples of ATTO dyes include, but are not limited to: ATTO
390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520,
ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B,
ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO
594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO
647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700,
ATTO 725, and ATTO 740.
[0462] Examples of AlexaFluor dyes include, but are not limited to:
Alexa Fluor.RTM. 350, Alexa Fluor.RTM. 405, Alexa Fluor.RTM. 430,
Alexa Fluor.RTM. 488, Alexa Fluor.RTM. 500, Alexa Fluor.RTM. 514,
Alexa Fluor.RTM. 532, Alexa Fluor.RTM. 546, Alexa Fluor.RTM. 555,
Alexa Fluor.RTM. 568, Alexa Fluor.RTM. 594, Alexa Fluor.RTM. 610,
Alexa Fluor.RTM. 633, Alexa Fluor.RTM. 635, Alexa Fluor.RTM. 647,
Alexa Fluor.RTM. 660, Alexa Fluor.RTM. 680, Alexa Fluor.RTM. 700,
Alexa Fluor.RTM. 750, Alexa Fluor.RTM. 790, and the like.
[0463] Examples of quencher moieties include, but are not limited
to: a dark quencher, a Black Hole Quencher.RTM. (BHQ.RTM.) (e.g.,
BHQ-0, BHQ-1, BHQ-2, BHQ-3), a Qxl quencher, an ATTO quencher
(e.g., ATTO 540Q, ATTO 580Q, and ATTO 612Q),
dimethylaminoazobenzenesulfonic acid (Dabsyl), Iowa Black RQ, Iowa
Black FQ, IRDye QC-1, a QSY dye (e.g., QSY 7, QSY 9, QSY 21),
AbsoluteQuencher, Eclipse, and metal clusters such as gold
nanoparticles, and the like.
[0464] In some cases, a quencher moiety is selected from: a dark
quencher, a Black Hole Quencher.RTM. (BHQ.RTM.) (e.g., BHQ-0,
BHQ-1, BHQ-2, BHQ-3), a Qxl quencher, an ATTO quencher (e.g., ATTO
540Q, ATTO 580Q, and ATTO 612Q), dimethylaminoazobenzenesulfonic
acid (Dabsyl), Iowa Black RQ, Iowa Black FQ, IRDye QC-1, a QSY dye
(e.g., QSY 7, QSY 9, QSY 21), AbsoluteQuencher, Eclipse, and a
metal cluster.
[0465] Examples of an ATTO quencher include, but are not limited
to: ATTO 540Q, ATTO 580Q, and ATTO 612Q. Examples of a Black Hole
Quencher.RTM. (BHQ.RTM.) include, but are not limited to: BHQ-0
(493 nm). BHQ-1 (534 nm), BHQ-2 (579 nm) and BHQ-3 (672 nm).
[0466] For examples of some detectable labels (e.g., fluorescent
dyes) and/or quencher moieties, see, e.g., Bao et al., Annu Rev
Biomed Eng. 2009; 11:25-47; as well as U.S. Pat. Nos. 8,822,673 and
8,586,718; U.S. patent publications 20140378330, 20140349295,
20140194611, 20130323851, 20130224871, 20110223677, 20110190486,
20110172420, 20060179585 and 20030003486; and international patent
applications: WO200142505 and WO200186001, all of which are hereby
incorporated by reference in their entirety.
[0467] In some cases, cleavage of a labeled detector ssDNA can be
detected by measuring a colorimetric read-out. For example, the
liberation of a fluorophore (e.g., liberation from a FRET pair,
liberation from a quencher/fluor pair, and the like) can result in
a wavelength shift (and thus color shift) of a detectable signal.
Thus, in some cases, cleavage of a subject labeled detector ssDNA
can be detected by a color-shift. Such a shift can be expressed as
a loss of an amount of signal of one color (wavelength), a gain in
the amount of another color, a change in the ration of one color to
another, and the like.
Transgenic, Non-Human Organisms
[0468] As described above, in some cases, a nucleic acid (e.g., a
recombinant expression vector) of the present disclosure (e.g., a
nucleic acid comprising a nucleotide sequence encoding a Cas12J
polypeptide of the present disclosure; a nucleic acid comprising a
nucleotide sequence encoding a Cas12J fusion polypeptide of the
present disclosure; etc.), is used as a transgene to generate a
transgenic non-human organism that produces a Cas12J polypeptide,
or a Cas12J fusion polypeptide, of the present disclosure. The
present disclosure provides a transgenic-non-human organism
comprising a nucleotide sequence encoding a Cas12J polypeptide, or
a Cas12J fusion polypeptide, of the present disclosure.
Transgenic, Non-Human Animals
[0469] The present disclosure provides a transgenic non-human
animal, which animal comprises a transgene comprising a nucleic
acid comprising a nucleotide sequence encoding a Cas12J polypeptide
or a Cas12J fusion polypeptide. In some embodiments, the genome of
the transgenic non-human animal comprises a nucleotide sequence
encoding a Cas12J polypeptide or a Cas12J fusion polypeptide, of
the present disclosure. In some cases, the transgenic non-human
animal is homozygous for the genetic modification. In some cases,
the transgenic non-human animal is heterozygous for the genetic
modification. In some embodiments, the transgenic non-human animal
is a vertebrate, for example, a fish (e.g., salmon, trout, zebra
fish, gold fish, puffer fish, cave fish, etc.), an amphibian (frog,
newt, salamander, etc.), a bird (e.g., chicken, turkey, etc.), a
reptile (e.g., snake, lizard, etc.), a non-human mammal (e.g., an
ungulate, e.g., a pig, a cow, a goat, a sheep, etc.; a lagomorph
(e.g., a rabbit); a rodent (e.g., a rat, a mouse); a non-human
primate; etc.), etc. In some cases, the transgenic non-human animal
is an invertebrate. In some cases, the transgenic non-human animal
is an insect (e.g., a mosquito; an agricultural pest; etc.). In
some cases, the transgenic non-human animal is an arachnid.
[0470] Nucleotide sequences encoding a a Cas12J polypeptide, e or a
Cas12J fusion polypeptide, of the present disclosure can be under
the control of (i.e., operably linked to) an unknown promoter
(e.g., when the nucleic acid randomly integrates into a host cell
genome) or can be under the control of (i.e., operably linked to) a
known promoter. Suitable known promoters can be any known promoter
and include constitutively active promoters (e.g., CMV promoter),
inducible promoters (e.g., heat shock promoter,
tetracycline-regulated promoter, steroid-regulated promoter,
metal-regulated promoter, estrogen receptor-regulated promoter,
etc.), spatially restricted and/or temporally restricted promoters
(e.g., a tissue specific promoter, a cell type specific promoter,
etc.), etc.
Transgenic Plants
[0471] As described above, in some cases, a nucleic acid (e.g., a
recombinant expression vector) of the present disclosure (e.g., a
nucleic acid comprising a nucleotide sequence encoding a Cas12J
polypeptide of the present disclosure; a nucleic acid comprising a
nucleotide sequence encoding a Cas12J fusion polypeptide of the
present disclosure; etc.), is used as a transgene to generate a
transgenic plant that produces a Cas12J polypeptide, or a Cas12J
fusion polypeptide, of the present disclosure. The present
disclosure provides a transgenic plant comprising a nucleotide
sequence encoding a Cas12J polypeptide, or a Cas12J fusion
polypeptide, of the present disclosure. In some embodiments, the
genome of the transgenic plant comprises a subject nucleic acid. In
some embodiments, the transgenic plant is homozygous for the
genetic modification. In some embodiments, the transgenic plant is
heterozygous for the genetic modification.
[0472] Methods of introducing exogenous nucleic acids into plant
cells are well known in the art. Such plant cells are considered
"transformed," as defined above. Suitable methods include viral
infection (such as double stranded DNA viruses), transfection,
conjugation, protoplast fusion, electroporation, particle gun
technology, calcium phosphate precipitation, direct microinjection,
silicon carbide whiskers technology, Agrobacterium-mediated
transformation and the like. The choice of method is generally
dependent on the type of cell being transformed and the
circumstances under which the transformation is taking place (i.e.
in vitro, ex vivo, or in vivo).
[0473] Transformation methods based upon the soil bacterium
Agrobacterium tumefaciens are particularly useful for introducing
an exogenous nucleic acid molecule into a vascular plant. The wild
type form of Agrobacterium contains a Ti (tumor-inducing) plasmid
that directs production of tumorigenic crown gall growth on host
plants. Transfer of the tumor-inducing T-DNA region of the Ti
plasmid to a plant genome requires the Ti plasmid-encoded virulence
genes as well as T-DNA borders, which are a set of direct DNA
repeats that delineate the region to be transferred. An
Agrobacterium-based vector is a modified form of a Ti plasmid, in
which the tumor inducing functions are replaced by the nucleic acid
sequence of interest to be introduced into the plant host.
[0474] Agrobacterium-mediated transformation generally employs
cointegrate vectors or binary vector systems, in which the
components of the Ti plasmid are divided between a helper vector,
which resides permanently in the Agrobacterium host and carries the
virulence genes, and a shuttle vector, which contains the gene of
interest bounded by T-DNA sequences. A variety of binary vectors is
well known in the art and are commercially available, for example,
from Clontech (Palo Alto, Calif.). Methods of coculturing
Agrobacterium with cultured plant cells or wounded tissue such as
leaf tissue, root explants, hypocotyledons, stem pieces or tubers,
for example, also are well known in the art. See, e.g., Glick and
Thompson, (eds.), Methods in Plant Molecular Biology and
Biotechnology, Boca Raton, Fla.: CRC Press (1993).
[0475] Microprojectile-mediated transformation also can be used to
produce a subject transgenic plant. This method, first described by
Klein et al. (Nature 327:70-73 (1987)), relies on microprojectiles
such as gold or tungsten that are coated with the desired nucleic
acid molecule by precipitation with calcium chloride, spermidine or
polyethylene glycol. The microprojectile particles are accelerated
at high speed into an angiosperm tissue using a device such as the
BIOLISTIC PD-1000 (Biorad; Hercules Calif.).
[0476] A nucleic acid of the present disclosure (e.g., a nucleic
acid (e.g., a recombinant expression vector) comprising a
nucleotide sequence encoding a Cas12J polypeptide, or a Cas12J
fusion polypeptide, of the present disclosure) may be introduced
into a plant in a manner such that the nucleic acid is able to
enter a plant cell(s), e.g., via an in vivo or ex vivo protocol. By
"in vivo," it is meant in the nucleic acid is administered to a
living body of a plant e.g. infiltration. By "ex vivo" it is meant
that cells or explants are modified outside of the plant, and then
such cells or organs are regenerated to a plant. A number of
vectors suitable for stable transformation of plant cells or for
the establishment of transgenic plants have been described,
including those described in Weissbach and Weissbach, (1989)
Methods for Plant Molecular Biology Academic Press, and Gelvin et
al., (1990) Plant Molecular Biology Manual, Kluwer Academic
Publishers. Specific examples include those derived from a Ti
plasmid of Agrobacterium tumefaciens, as well as those disclosed by
Herrera-Estrella et al. (1983) Nature 303: 209, Bevan (1984) Nucl
Acid Res. 12: 8711-8721, Klee (1985) Bio/Technolo 3: 637-642.
Alternatively, non-Ti vectors can be used to transfer the DNA into
plants and cells by using free DNA delivery techniques. By using
these methods transgenic plants such as wheat, rice (Christou
(1991) Bio/Technology 9:957-9 and 4462) and corn (Gordon-Kamm
(1990) Plant Cell 2: 603-618) can be produced. An immature embryo
can also be a good target tissue for monocots for direct DNA
delivery techniques by using the particle gun (Weeks et al. (1993)
Plant Physiol 102: 1077-1084; Vasil (1993) Bio/Technolo 10:
667-674; Wan and Lemeaux (1994) Plant Physiol 104: 37-48 and for
Agrobacterium-mediated DNA transfer (Ishida et al. (1996) Nature
Biotech 14: 745-750). Exemplary methods for introduction of DNA
into chloroplasts are biolistic bombardment, polyethylene glycol
transformation of protoplasts, and microinjection (Danieli et al
Nat. Biotechnol 16:345-348, 1998; Staub et al Nat. Biotechnol 18:
333-338, 2000; O'Neill et al Plant J. 3:729-738, 1993; Knoblauch et
al Nat. Biotechnol 17: 906-909; U.S. Pat. Nos. 5,451,513,
5,545,817, 5,545,818, and 5,576,198; in Intl. Application No. WO
95/16783; and in Boynton et al., Methods in Enzymology 217: 510-536
(1993), Svab et al., Proc. Natl. Acad. Sci. USA 90: 913-917 (1993),
and McBride et al., Proc. Natl. Acad. Sci. USA 91: 7301-7305
(1994)). Any vector suitable for the methods of biolistic
bombardment, polyethylene glycol transformation of protoplasts and
microinjection will be suitable as a targeting vector for
chloroplast transformation. Any double stranded DNA vector may be
used as a transformation vector, especially when the method of
introduction does not utilize Agrobacterium.
[0477] Plants which can be genetically modified include grains,
forage crops, fruits, vegetables, oil seed crops, palms, forestry,
and vines. Specific examples of plants which can be modified
follow: maize, banana, peanut, field peas, sunflower, tomato,
canola, tobacco, wheat, barley, oats, potato, soybeans, cotton,
carnations, sorghum, lupin and rice.
[0478] The present disclosure provides transformed plant cells,
tissues, plants and products that contain the transformed plant
cells. A feature of the subject transformed cells, and tissues and
products that include the same is the presence of a subject nucleic
acid integrated into the genome, and production by plant cells of a
Cas12J polypeptide, or a Cas12J fusion polypeptide, of the present
disclosure. Recombinant plant cells of the present invention are
useful as populations of recombinant cells, or as a tissue, seed,
whole plant, stem, fruit, leaf, root, flower, stem, tuber, grain,
animal feed, a field of plants, and the like.
[0479] Nucleotide sequences encoding a Cas12J polypeptide, or a
Cas12J fusion polypeptide, of the present disclosure can be under
the control of (i.e., operably linked to) an unknown promoter
(e.g., when the nucleic acid randomly integrates into a host cell
genome) or can be under the control of (i.e., operably linked to) a
known promoter. Suitable known promoters can be any known promoter
and include constitutively active promoters, inducible promoters,
spatially restricted and/or temporally restricted promoters,
etc.
Examples of Non-Limiting Aspects of the Disclosure
[0480] Aspects, including embodiments, of the present subject
matter described above may be beneficial alone or in combination,
with one or more other aspects or embodiments. Without limiting the
foregoing description, certain non-limiting aspects of the
disclosure numbered 1-149 are provided below. As will be apparent
to those of skill in the art upon reading this disclosure, each of
the individually numbered aspects may be used or combined with any
of the preceding or following individually numbered aspects. This
is intended to provide support for all such combinations of aspects
and is not limited to combinations of aspects explicitly provided
below:
[0481] Aspect 1. A composition comprising: a) a Cas12J polypeptide,
or a nucleic acid molecule encoding the Cas12J polypeptide; and b)
a Cas12J guide RNA, or one or more DNA molecules encoding the
Cas12J guide RNA.
[0482] Aspect 2. The composition of aspect 1, wherein the Cas12J
polypeptide comprises an amino acid sequence having 50% or more
amino acid sequence identity to the amino acid sequence depicted in
any one of FIG. 6A-6R.
[0483] Aspect 3. The composition of aspect 1 or aspect 2, wherein
the Cas12J guide RNA comprises a nucleotide sequence having 80%,
90%, 95%, 98%, 99%, or 100%, nucleotide sequence identity with any
one of the crRNA sequences depicted in FIG. 7.
[0484] Aspect 4. The composition of aspect 1 or aspect 2, wherein
the Cas12J polypeptide is fused to a nuclear localization signal
(NLS).
[0485] Aspect 5. The composition of any one of aspects 1-4, wherein
the composition comprises a lipid.
[0486] Aspect 6. The composition of any one of aspects 1-4, wherein
a) and b) are within a liposome.
[0487] Aspect 7. The composition of any one of aspects 1-4, wherein
a) and b) are within a particle.
[0488] Aspect 8. The composition of any one of aspects 1-7,
comprising one or more of: a buffer, a nuclease inhibitor, and a
protease inhibitor.
[0489] Aspect 9. The composition of any one of aspects 1-8, wherein
the Cas12J polypeptide comprises an amino acid sequence having 85%
or more identity to the amino acid sequence depicted in any one of
FIG. 6A-6R.
[0490] Aspect 10. The composition of any one of aspects 1-9,
wherein the Cas12J polypeptide is a nickase that can cleave only
one strand of a double-stranded target nucleic acid molecule.
[0491] Aspect 11. The composition of any one of aspects 1-9,
wherein the Cas12J polypeptide is a catalytically inactive Cas12J
polypeptide (dCas12J).
[0492] Aspect 12. The composition of aspect 10 or aspect 11,
wherein the Cas12J polypeptide comprises one or more mutations at a
position corresponding to those selected from: D464, E678, and D769
of Cas12J_10037042_3.
[0493] Aspect 13. The composition of any one of aspects 1-12,
further comprising a DNA donor template.
[0494] Aspect 14. A Cas12J fusion polypeptide comprising: a Cas12J
polypeptide fused to a heterologous polypeptide.
[0495] Aspect 15, The Cas12J fusion polypeptide of Aspect 14,
wherein the Cas12J polypeptide comprises an amino acid sequence
having 50% or more identity to the amino acid sequence depicted in
any one of FIG. 6A-6R.
[0496] Aspect 16. The Cas12J fusion polypeptide of Aspect 14,
wherein the Cas12J polypeptide comprises an amino acid sequence
having 85% or more identity to the amino acid sequence depicted in
any one of FIG. 6A-6R.
[0497] Aspect 17. The Cas12J fusion polypeptide of any one of
aspects 14-16, wherein the Cas12J polypeptide is a nickase that can
cleave only one strand of a double-stranded target nucleic acid
molecule.
[0498] Aspect 18. The Cas12J fusion polypeptide of any one of
aspects 14-17, wherein the Cas12J polypeptide is a catalytically
inactive Cas12J polypeptide (dCas12J).
[0499] Aspect 19. The Cas12J fusion polypeptide of aspect 17 or
aspect 18, wherein the Cas12J polypeptide comprises one or more
mutations at a position corresponding to those selected from: D464,
E678, and D769 of Cas12L10037042_3.
[0500] Aspect 20. The Cas12J fusion polypeptide of any one of
aspects 14-19, wherein the heterologous polypeptide is fused to the
N-terminus and/or the C-terminus of the Cas12J polypeptide.
[0501] Aspect 21. The Cas12J fusion polypeptide of any one of
aspects 14-20, comprising a nuclear localization signal (NLS).
[0502] Aspect 22. The Cas12J fusion polypeptide of any one of
aspects 14-21, wherein the heterologous polypeptide is a targeting
polypeptide that provides for binding to a cell surface moiety on a
target cell or target cell type.
[0503] Aspect 23. The Cas12J fusion polypeptide of any one of
aspects 14-21, wherein the heterologous polypeptide exhibits an
enzymatic activity that modifies target DNA.
[0504] Aspect 24. The Cas12J fusion polypeptide of aspect 23,
wherein the heterologous polypeptide exhibits one or more enzymatic
activities selected from: nuclease activity, methyltransferase
activity, demethylase activity, DNA repair activity, DNA damage
activity, deamination activity, dismutase activity, alkylation
activity, depurination activity, oxidation activity, pyrimidine
dimer forming activity, integrase activity, transposase activity,
recombinase activity, polymerase activity, ligase activity,
helicase activity, photolyase activity and glycosylase
activity.
[0505] Aspect 25. The Cas12J fusion polypeptide of aspect 24,
wherein the heterologous polypeptide exhibits one or more enzymatic
activities selected from: nuclease activity, methyltransferase
activity, demethylase activity, deamination activity, depurination
activity, integrase activity, transposase activity, and recombinase
activity.
[0506] Aspect 26. The Cas12J fusion polypeptide of any one of
aspects 14-21, wherein the heterologous polypeptide exhibits an
enzymatic activity that modifies a target polypeptide associated
with a target nucleic acid.
[0507] Aspect 27. The Cas12J fusion polypeptide of aspect 26,
wherein the heterologous polypeptide exhibits histone modification
activity.
[0508] Aspect 28. The Cas12J fusion polypeptide of aspect 26 or
aspect 27, wherein the heterologous polypeptide exhibits one or
more enzymatic activities selected from: methyltransferase
activity, demethylase activity, acetyltransferase activity,
deacetylase activity, kinase activity, phosphatase activity,
ubiquitin ligase activity, deubiquitinating activity, adenylation
activity, deadenylation activity, SUMOylating activity,
deSUMOylating activity, ribosylation activity, deribosylation
activity, myristoylation activity, demyristoylation activity,
glycosylation activity (e.g., from O-GlcNAc transferase) and
deglycosylation activity.
[0509] Aspect 29. The Cas12J fusion polypeptide of aspect 28,
wherein the heterologous polypeptide exhibits one or more enzymatic
activities selected from: methyltransferase activity, demethylase
activity, acetyltransferase activity, and deacetylase activity.
[0510] Aspect 30. The Cas12J fusion polypeptide of any one of
aspects 14-21, wherein the heterologous polypeptide is an endosomal
escape polypeptide.
[0511] Aspect 31. The Cas12J fusion polypeptide of aspect 30,
wherein the endosomal escape polypeptide comprises an amino acid
sequence selected from: GLFXALLXLLXSLWXLLLXA (SEQ ID NO: 36), and
GLFHALLHLLHSLWHLLLHA (SEQ ID NO: 37), wherein each X is
independently selected from lysine, histidine, and arginine.
[0512] Aspect 32. The Cas12J fusion polypeptide of any one of
aspects 14-21, wherein the heterologous polypeptide is a
chloroplast transit peptide.
[0513] Aspect 33. The Cas12J fusion polypeptide of any one of
aspects 14-21, wherein the heterologous polypeptide comprises a
protein transduction domain.
[0514] Aspect 34. The Cas12J fusion polypeptide of any one of
aspects 14-21, wherein the heterologous polypeptide is a protein
that increases or decreases transcription.
[0515] Aspect 35. The Cas12J fusion polypeptide of aspect 34,
wherein the heterologous polypeptide is a transcriptional repressor
domain.
[0516] Aspect 36. The Cas12J fusion polypeptide of aspect 34,
wherein the heterologous polypeptide is a transcriptional
activation domain.
[0517] Aspect 37. The Cas12J fusion polypeptide of any one of
aspects 14-21, wherein the heterologous polypeptide is a protein
binding domain.
[0518] Aspect 38. A nucleic acid comprising a nucleotide sequence
encoding the Cas12J fusion polypeptide of any one of aspects
14-37.
[0519] Aspect 39. The nucleic acid of Aspect 38, wherein the
nucleotide sequence encoding the Cas12J fusion polypeptide is
operably linked to a promoter.
[0520] Aspect 40. The nucleic acid of Aspect 39, wherein the
promoter is functional in a eukaryotic cell.
[0521] Aspect 41. The nucleic acid of Aspect 40, wherein the
promoter is functional in one or more of: a plant cell, a fungal
cell, an animal cell, cell of an invertebrate, a fly cell, a cell
of a vertebrate, a mammalian cell, a primate cell, a non-human
primate cell, and a human cell.
[0522] Aspect 43. The nucleic acid of any one of Aspects 39-41,
wherein the promoter is one or more of: a constitutive promoter, an
inducible promoter, a cell type-specific promoter, and a
tissue-specific promoter.
[0523] Aspect 43. The nucleic acid of any one of Aspects 38-42,
wherein the nucleic acid is a recombinant expression vector.
[0524] Aspect 44. The nucleic acid of Aspect 43, wherein the
recombinant expression vector is a recombinant adenoassociated
viral vector, a recombinant retroviral vector, or a recombinant
lentiviral vector.
[0525] Aspect 45. The nucleic acid of Aspect 39, wherein the
promoter is functional in a prokaryotic cell.
[0526] Aspect 46. The nucleic acid of Aspect 38, wherein the
nucleic acid molecule is an mRNA.
[0527] Aspect 47. One or more nucleic acids comprising: (a) a
nucleotide sequence encoding a Cas12J guide RNA; and (b) a
nucleotide sequence encoding a Cas12J polypeptide.
[0528] Aspect 48. The one or more nucleic acids of aspect 47,
wherein the Cas12J polypeptide comprises an amino acid sequence
having 50% or more identity to the amino acid sequence depicted in
any one of FIG. 6A-6R.
[0529] Aspect 49. The one or more nucleic acids of aspect 47,
wherein the Cas12J polypeptide comprises an amino acid sequence
having 85% or more identity to the amino acid depicted in any one
of FIG. 6A-6R.
[0530] Aspect 50. The one or more nucleic acids of any one of
aspects 47-49, wherein the Cas12J guide RNA comprises a nucleotide
sequence having 80% or more nucleotide sequence identity with any
one of the crRNA sequences set forth in FIG. 7.
[0531] Aspect 51. The one or more nucleic acids of any one of
aspects 47-50, wherein the Cas12J polypeptide is fused to a nuclear
localization signal (NLS).
[0532] Aspect 52. The one or more nucleic acids of any one of
aspects 47-51, wherein the nucleotide sequence encoding the Cas12J
guide RNA is operably linked to a promoter.
[0533] Aspect 53. The one or more nucleic acids of any one of
aspects 47-52, wherein the nucleotide sequence encoding the Cas12J
polypeptide is operably linked to a promoter.
[0534] Aspect 54. The one or more nucleic acids of Aspect 52 or
Aspect 53, wherein the promoter operably linked to the nucleotide
sequence encoding the Cas12J guide RNA, and/or the promoter
operably linked to the nucleotide sequence encoding the Cas12J
polypeptide, is functional in a eukaryotic cell.
[0535] Aspect 55. The one or more nucleic acids of Aspect 54,
wherein the promoter is functional in one or more of: a plant cell,
a fungal cell, an animal cell, cell of an invertebrate, a fly cell,
a cell of a vertebrate, a mammalian cell, a primate cell, a
non-human primate cell, and a human cell.
[0536] Aspect 56. The one or more nucleic acids of any one of
Aspects 53-55, wherein the promoter is one or more of: a
constitutive promoter, an inducible promoter, a cell type-specific
promoter, and a tissue-specific promoter.
[0537] Aspect 57. The one or more nucleic acids of any one of
Aspects 47-56, wherein the one or more nucleic acids is one or more
recombinant expression vectors.
[0538] Aspect 58. The one or more nucleic acids of Aspect 57,
wherein the one or more recombinant expression vectors are selected
from: one or more adenoassociated viral vectors, one or more
recombinant retroviral vectors, or one or more recombinant
lentiviral vectors.
[0539] Aspect 59. The one or more nucleic acids of Aspect 53,
wherein the promoter is functional in a prokaryotic cell.
[0540] Aspect 60. A eukaryotic cell comprising one or more of: a) a
Cas12J polypeptide, or a nucleic acid comprising a nucleotide
sequence encoding the Cas12J polypeptide, b) a Cas12J fusion
polypeptide, or a nucleic acid comprising a nucleotide sequence
encoding the Cas12J fusion polypeptide, and c) a Cas12J guide RNA,
or a nucleic acid comprising a nucleotide sequence encoding the
Cas12J guide RNA.
[0541] Aspect 61. The eukaryotic cell of aspect 60, comprising the
nucleic acid encoding the Cas12J polypeptide, wherein said nucleic
acid is integrated into the genomic DNA of the cell.
[0542] Aspect 62. The eukaryotic cell of aspect 60 or aspect 61,
wherein the eukaryotic cell is a plant cell, a mammalian cell, an
insect cell, an arachnid cell, a fungal cell, a bird cell, a
reptile cell, an amphibian cell, an invertebrate cell, a mouse
cell, a rat cell, a primate cell, a non-human primate cell, or a
human cell.
[0543] Aspect 63. A cell comprising a comprising a Cas12J fusion
polypeptide, or a nucleic acid comprising a nucleotide sequence
encoding the Cas12J fusion polypeptide.
[0544] Aspect 64. The cell of aspect 63, wherein the cell is a
prokaryotic cell.
[0545] Aspect 65. The cell of aspect 63 or aspect 64, comprising
the nucleic acid comprising a nucleotide sequence encoding the
Cas12J fusion polypeptide, wherein said nucleic acid molecule is
integrated into the genomic DNA of the cell.
[0546] Aspect 66. A method of modifying a target nucleic acid, the
method comprising contacting the target nucleic acid with: a) a
Cas12J polypeptide; and b) a Cas12J guide RNA comprising a guide
sequence that hybridizes to a target sequence of the target nucleic
acid, wherein said contacting results in modification of the target
nucleic acid by the Cas12J polypeptide.
[0547] Aspect 67. The method of aspect 66, wherein said
modification is cleavage of the target nucleic acid.
[0548] Aspect 68. The method of aspect 66 or aspect 67, wherein the
target nucleic acid is selected from: double stranded DNA, single
stranded DNA, RNA, genomic DNA, and extrachromosomal DNA.
[0549] Aspect 69. The method of any of aspects 66-68, wherein said
contacting takes place in vitro outside of a cell.
[0550] Aspect 70. The method of any of aspects 66-68, wherein said
contacting takes place inside of a cell in culture.
[0551] Aspect 71. The method of any of aspects 66-68, wherein said
contacting takes place inside of a cell in vivo.
[0552] Aspect 72. The method of aspect 70 or aspect 71, wherein the
cell is a eukaryotic cell.
[0553] Aspect 73. The method of aspect 72, wherein the cell is
selected from: a plant cell, a fungal cell, a mammalian cell, a
reptile cell, an insect cell, an avian cell, a fish cell, a
parasite cell, an arthropod cell, a cell of an invertebrate, a cell
of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate
cell, a non-human primate cell, and a human cell.
[0554] Aspect 74. The method of aspect 70 or aspect 71, wherein the
cell is a prokaryotic cell.
[0555] Aspect 75. The method of any one of aspects 66-74, wherein
said contacting results in genome editing.
[0556] Aspect 76. The method of any one of aspects 66-75, wherein
said contacting comprises: introducing into a cell: (a) the Cas12J
polypeptide, or a nucleic acid comprising a nucleotide sequence
encoding the Cas12J polypeptide, and (b) the Cas12J guide RNA, or a
nucleic acid comprising a nucleotide sequence encoding the Cas12J
guide RNA.
[0557] Aspect 77. The method of aspect 76, wherein said contacting
further comprises: introducing a DNA donor template into the
cell.
[0558] Aspect 78. The method of any one of aspects 66-77, wherein
the Cas12J guide RNA comprises a nucleotide sequence having 80% or
more nucleotide sequence identity with any one of the crRNA
sequences set forth in FIG. 7.
[0559] Aspect 79. The method of any one of aspects 66-78, wherein
the Cas12J polypeptide is fused to a nuclear localization
signal.
[0560] Aspect 80. A method of modulating transcription from a
target DNA, modifying a target nucleic acid, or modifying a protein
associated with a target nucleic acid, the method comprising
contacting the target nucleic acid with: a) a Cas12J fusion
polypeptide comprising a Cas12J polypeptide fused to a heterologous
polypeptide; and b) a Cas12J guide RNA comprising a guide sequence
that hybridizes to a target sequence of the target nucleic
acid.
[0561] Aspect 81. The method of aspect 80, wherein the Cas12J guide
RNA comprises a nucleotide sequence having 80% or more nucleotide
sequence identity with any one of the crRNA sequences set forth in
FIG. 7.
[0562] Aspect 82. The method of aspect 80 or aspect 81, wherein the
Cas12J fusion polypeptide comprises nuclear localization
signal.
[0563] Aspect 83. The method of any of aspects 80-82, wherein said
modification is not cleavage of the target nucleic acid.
[0564] Aspect 84. The method of any of aspects 80-83, wherein the
target nucleic acid is selected from: double stranded DNA, single
stranded DNA, RNA, genomic DNA, and extrachromosomal DNA.
[0565] Aspect 85. The method of any of aspects 80-84, wherein said
contacting takes place in vitro outside of a cell.
[0566] Aspect 86. The method of any of aspects 80-84, wherein said
contacting takes place inside of a cell in culture.
[0567] Aspect 87. The method of any of aspects 80-84, wherein said
contacting takes place inside of a cell in vivo.
[0568] Aspect 88. The method of aspect 86 or aspect 87, wherein the
cell is a eukaryotic cell.
[0569] Aspect 89. The method of aspect 88, wherein the cell is
selected from: a plant cell, a fungal cell, a mammalian cell, a
reptile cell, an insect cell, an avian cell, a fish cell, a
parasite cell, an arthropod cell, a cell of an invertebrate, a cell
of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate
cell, a non-human primate cell, and a human cell.
[0570] Aspect 90. The method of aspect 86 or aspect 87, wherein the
cell is a prokaryotic cell.
[0571] Aspect 91. The method of any one of aspects 80-90, wherein
said contacting comprises: introducing into a cell: (a) the Cas12J
fusion polypeptide, or a nucleic acid comprising a nucleotide
sequence encoding the Cas12J fusion polypeptide, and (b) the Cas12J
guide RNA, or a nucleic acid comprising a nucleotide sequence
encoding the Cas12J guide RNA.
[0572] Aspect 92. The method of any one of aspects 80-91, wherein
the Cas12J polypeptide is a catalytically inactive Cas12J
polypeptide (dCas12J).
[0573] Aspect 93. The method of any one of aspects 80-92, wherein
the Cas12J polypeptide comprises one or more amino acid
substitutions at a position corresponding to those selected from:
D464, E678, and D769 of Cas12J_10037042_3.
[0574] Aspect 94. The method of any one of aspects 80-93, wherein
the heterologous polypeptide exhibits an enzymatic activity that
modifies target DNA.
[0575] Aspect 95. The method of aspect 94, wherein the heterologous
polypeptide exhibits an one or more enzymatic activities selected
from: nuclease activity, methyltransferase activity, demethylase
activity, DNA repair activity, DNA damage activity, deamination
activity, dismutase activity, alkylation activity, depurination
activity, oxidation activity, pyrimidine dimer forming activity,
integrase activity, transposase activity, recombinase activity,
polymerase activity, ligase activity, helicase activity, photolyase
activity and glycosylase activity.
[0576] Aspect 96. The method of aspect 95, wherein the heterologous
polypeptide exhibits one or more enzymatic activities selected
from: nuclease activity, methyltransferase activity, demethylase
activity, deamination activity, depurination activity, integrase
activity, transposase activity, and recombinase activity.
[0577] Aspect 97. The method of any one of aspects 80-93, wherein
the heterologous polypeptide exhibits an enzymatic activity that
modifies a target polypeptide associated with a target nucleic
acid.
[0578] Aspect 98. The method of aspect 97, wherein the heterologous
polypeptide exhibits histone modification activity.
[0579] Aspect 99. The method of aspect 97 or aspect 98, wherein the
heterologous polypeptide exhibits an one or more enzymatic
activities selected from: methyltransferase activity, demethylase
activity, acetyltransferase activity, deacetylase activity, kinase
activity, phosphatase activity, ubiquitin ligase activity,
deubiquitinating activity, adenylation activity, deadenylation
activity, SUMOylating activity, deSUMOylating activity,
ribosylation activity, deribosylation activity, myristoylation
activity, demyristoylation activity, glycosylation activity (e.g.,
from 0-GlcNAc transferase) and deglycosylation activity.
[0580] Aspect 100. The method of aspect 99, wherein the
heterologous polypeptide exhibits one or more enzymatic activities
selected from: methyltransferase activity, demethylase activity,
acetyltransferase activity, and deacetylase activity.
[0581] Aspect 101. The method of any one of aspects 80-93, wherein
the heterologous polypeptide is protein that increases or decreases
transcription.
[0582] Aspect 102. The method of aspect 101, wherein the
heterologous polypeptide is a transcriptional repressor domain.
[0583] Aspect 103. The method of aspect 101, wherein the
heterologous polypeptide is a transcriptional activation
domain.
[0584] Aspect 104. The method of any one of aspects 80-93, wherein
the heterologous polypeptide is a protein binding domain.
[0585] Aspect 105. A transgenic, multicellular, non-human organism
whose genome comprises a transgene comprising a nucleotide sequence
encoding one or more of: a) a Cas12J polypeptide; b) a Cas12J
fusion polypeptide; and c) a Cas12J guide RNA
[0586] Aspect 106. The transgenic, multicellular, non-human
organism of aspect 105, wherein the Cas12J polypeptide comprises an
amino acid sequence having 50% or more amino acid sequence identity
to the amino acid sequence set forth in any one of FIG. 6A-6R.
[0587] Aspect 107. The transgenic, multicellular, non-human
organism of aspect 105, wherein the Cas12J polypeptide comprises an
amino acid sequence having 85% or more amino acid sequence identity
to the amino acid sequence set forth in any one of FIG. 6A-6R.
[0588] Aspect 108. The transgenic, multicellular, non-human
organism of any one of aspects 105-107, wherein the organism is a
plant, a monocotyledon plant, a dicotyledon plant, an invertebrate
animal, an insect, an arthropod, an arachnid, a parasite, a worm, a
cnidarian, a vertebrate animal, a fish, a reptile, an amphibian, an
ungulate, a bird, a pig, a horse, a sheep, a rodent, a mouse, a
rat, or a non-human primate.
[0589] Aspect 109. A system comprising one of:
[0590] a) a Cas12J polypeptide and a Cas12J guide RNA;
[0591] b) a Cas12J polypeptide, a Cas12J guide RNA, and a DNA donor
template;
[0592] c) a Cas12J fusion polypeptide and a Cas12J guide RNA;
[0593] d) a Cas12J fusion polypeptide, a Cas12J guide RNA, and a
DNA donor template;
[0594] e) an mRNA encoding a Cas12J polypeptide, and a Cas12J guide
RNA;
[0595] f) an mRNA encoding a Cas12J polypeptide; a Cas12J guide
RNA, and a DNA donor template;
[0596] g) an mRNA encoding a Cas12J fusion polypeptide, and a
Cas12J guide RNA;
[0597] h) an mRNA encoding a Cas12J fusion polypeptide, a Cas12J
guide RNA, and a DNA donor template;
[0598] i) one or more recombinant expression vectors comprising: i)
a nucleotide sequence encoding a Cas12J polypeptide; and ii) a
nucleotide sequence encoding a Cas12J guide RNA;
[0599] j) one or more recombinant expression vectors comprising: i)
a nucleotide sequence encoding a Cas12J polypeptide; ii) a
nucleotide sequence encoding a Cas12J guide RNA; and iii) a DNA
donor template;
[0600] k) one or more recombinant expression vectors comprising: i)
a nucleotide sequence encoding a Cas12J fusion polypeptide; and ii)
a nucleotide sequence encoding a Cas12J guide RNA; and
[0601] l) one or more recombinant expression vectors comprising: i)
a nucleotide sequence encoding a Cas12J fusion polypeptide; ii) a
nucleotide sequence encoding a Cas12J guide RNA; and a DNA donor
template.
[0602] Aspect 110. The Cas12J system of aspect 109, wherein the
Cas12J polypeptide comprises an amino acid sequence having 50% or
more amino acid sequence identity to the amino acid sequence
depicted in any one of FIG. 6A-6R.
[0603] Aspect 111. The Cas12J system of aspect 109, wherein the
Cas12J polypeptide comprises an amino acid sequence having 85% or
more amino acid sequence identity to the amino acid sequence
depicted in any one of FIG. 6A-6R.
[0604] Aspect 112. The Cas12J system of any of aspects 109-111,
wherein the donor template nucleic acid has a length of from 8
nucleotides to 1000 nucleotides.
[0605] Aspect 113. The Cas12J system of any of aspects 109-111,
wherein the donor template nucleic acid has a length of from 25
nucleotides to 500 nucleotides.
[0606] Aspect 114. A kit comprising the Cas12J system of any one of
aspects 109-113.
[0607] Aspect 115. The kit of aspect 114, wherein the components of
the kit are in the same container.
[0608] Aspect 116. The kit of aspect 114, wherein the components of
the kit are in separate containers.
[0609] Aspect 117. A sterile container comprising the Cas12J system
of any one of aspects 109-116.
[0610] Aspect 118. The sterile container of aspect 117, wherein the
container is a syringe.
[0611] Aspect 119. An implantable device comprising the Cas12J
system of any one of aspects 109-116.
[0612] Aspect 120. The implantable device of aspect 119, wherein
the Cas12J system is within a matrix.
[0613] Aspect 121. The implantable device of aspect 119, wherein
the Cas12J system is in a reservoir.
[0614] Aspect 122. A method of detecting a target DNA in a sample,
the method comprising: (a) contacting the sample with: (i) a Cas12L
polypeptide; (ii) a guide RNA comprising: a region that binds to
the Cas12L polypeptide, and a guide sequence that hybridizes with
the target DNA; and (iii) a detector DNA that is single stranded
and does not hybridize with the guide sequence of the guide RNA;
and (b) measuring a detectable signal produced by cleavage of the
single stranded detector DNA by the Cas12L polypeptide, thereby
detecting the target DNA.
[0615] Aspect 123. The method of aspect 122, wherein the target DNA
is single stranded.
[0616] Aspect 124. The method of aspect 122, wherein the target DNA
is double stranded.
[0617] Aspect 125. The method of any one of aspects 122-124,
wherein the target DNA is bacterial DNA.
[0618] Aspect 126. The method of any one of aspects 122-124,
wherein the target DNA is viral DNA.
[0619] Aspect 127. The method of aspect 126, wherein the target DNA
is papovavirus, human papillomavirus (HPV), hepadnavirus, Hepatitis
B Virus (HBV), herpesvirus, varicella zoster virus (VZV),
Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus,
adenovirus, poxvirus, or parvovirus DNA.
[0620] Aspect 128. The method of aspect 122, wherein the target DNA
is from a human cell.
[0621] Aspect 129. The method of aspect 122, wherein the target DNA
is human fetal or cancer cell DNA.
[0622] Aspect 130. The method of any one of aspects 122-129,
wherein the Cas12J polypeptide comprises an amino acid sequence
having 50% or more amino acid sequence identity to the amino acid
sequence depicted in any one of FIG. 6A-6R.
[0623] Aspect 131. The method of aspect 122, wherein the sample
comprises DNA from a cell lysate.
[0624] Aspect 132. The method of aspect 122, wherein the sample
comprises cells.
[0625] Aspect 133. The method of aspect 122, wherein the sample is
a blood, serum, plasma, urine, aspirate, or biopsy sample.
[0626] Aspect 134. The method of any one of aspects 122-133,
further comprising determining an amount of the target DNA present
in the sample.
[0627] Aspect 135. The method of aspect 122, wherein said measuring
a detectable signal comprises one or more of: visual based
detection, sensor-based detection, color detection, gold
nanoparticle based detection, fluorescence polarization, colloid
phase transition/dispersion, electrochemical detection, and
semiconductor-based sensing.
[0628] Aspect 136. The method of any one of aspects 122-135,
wherein the labeled detector DNA comprises a modified nucleobase, a
modified sugar moiety, and/or a modified nucleic acid linkage.
[0629] Aspect 137. The method of any one of aspects 122-135,
further comprising detecting a positive control target DNA in a
positive control sample, the detecting comprising: (c) contacting
the positive control sample with: (i) the Cas12J polypeptide; (ii)
a positive control guide RNA comprising: a region that binds to the
Cas12J polypeptide, and a positive control guide sequence that
hybridizes with the positive control target DNA; and (iii) a
labeled detector DNA that is single stranded and does not hybridize
with the positive control guide sequence of the positive control
guide RNA; and (d) measuring a detectable signal produced by
cleavage of the labeled detector DNA by the Cas12J polypeptide,
thereby detecting the positive control target DNA
[0630] Aspect 138. The method of any one of aspects 122-136,
wherein the detectable signal is detectable in less than 45
minutes.
[0631] Aspect 139. The method of any one of aspects 122-136,
wherein the detectable signal is detectable in less than 30
minutes.
[0632] Aspect 140. The method of any one of aspects 122-139,
further comprising amplifying the target DNA in the sample by
loop-mediated isothermal amplification (LAMP), helicase-dependent
amplification (HDA), recombinase polymerase amplification (RPA),
strand displacement amplification (SDA), nucleic acid
sequence-based amplification (NASBA), transcription mediated
amplification (TMA), nicking enzyme amplification reaction (NEAR),
rolling circle amplification (RCA), multiple displacement
amplification (MDA), Ramification (RAM), circular
helicase-dependent amplification (cHDA), single primer isothermal
amplification (SPIA), signal mediated amplification of RNA
technology (SMART), self-sustained sequence replication (3SR),
genome exponential amplification reaction (GEAR), or isothermal
multiple displacement amplification (IMDA).
[0633] Aspect 141. The method of any one of aspects 122-140,
wherein target DNA in the sample is present at a concentration of
less than 10 aM.
[0634] Aspect 142. The method according to any one of aspect
122-141, wherein the single stranded detector DNA comprises a
fluorescence-emitting dye pair.
[0635] Aspect 143. The method according to aspect 142, wherein the
fluorescence-emitting dye pair produces an amount of detectable
signal prior to cleavage of the single stranded detector DNA, and
the amount of detectable signal is reduced after cleavage of the
single stranded detector DNA.
[0636] Aspect 144. The method according to aspect 142, wherein the
single stranded detector DNA produces a first detectable signal
prior to being cleaved and a second detectable signal after
cleavage of the single stranded detector DNA.
[0637] Aspect 145. The method according to any one of aspects
142-144, wherein the fluorescence-emitting dye pair is a
fluorescence resonance energy transfer (FRET) pair.
[0638] Aspect 146. The method according to aspect 142, wherein an
amount of detectable signal increases after cleavage of the single
stranded detector DNA.
[0639] Aspect 147. The method according to any one of aspects
142-146, wherein the fluorescence-emitting dye pair is a
quencher/fluor pair.
[0640] Aspect 148. The method according to any one of aspects
142-147, wherein the single stranded detector DNA comprises two or
more fluorescence-emitting dye pairs.
[0641] Aspect 149. The method according to aspect 148, wherein said
two or more fluorescence-emitting dye pairs include a fluorescence
resonance energy transfer (FRET) pair and a quencher/fluor
pair.
EXAMPLES
[0642] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure
is at or near atmospheric. Standard abbreviations may be used,
e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or
sec, second(s); min, minute(s); h or hr, hour(s); aa, amino
acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s);
i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,
subcutaneous(ly); and the like.
Example 1
[0643] Metagenomic datasets from many diverse ecosystems were
generated and hundreds of huge phage genomes, between 200 kbp and
716 kbp in length, were reconstructed. Thirty-four genomes were
manually curated to completion, including the largest phage genomes
yet reported. Expanded genetic repertoires include diverse and new
CRISPR-Cas systems, tRNAs, tRNA synthetases, tRNA modification
enzymes, initiation and elongation factors and ribosomal proteins.
Phage CRISPR have the capacity to silence host transcription
factors and translational genes, potentially as part of a larger
interaction network that intercepts translation to redirect
biosynthesis to phage-encoded functions. Some phage repurpose
bacterial systems for phage-defense to eliminate competing phage.
Seven major clades of huge phage from human and other animal
microbiomes, oceans, lakes, sediments, soils and the built
environment were phylogenetic ally defined. It is concluded that
large gene inventories reflect a conserved biological strategy,
observed across a broad bacterial host range and resulting in the
distribution of huge phage across Earth's ecosystems.
[0644] Hundreds of phage sequences >200 kbp in length that were
reconstructed from microbiome datasets generated from a wide
variety of ecosystems were presented. The three largest complete
genomes for phage known to date, ranging up to 642 kbp in length,
were reconstructed. A graphical abstract provides an overview of
the approach and main findings. The research expands the
understanding of phage biodiversity and brings to light the variety
of ecosystems in which phage have genome sizes that rival those of
small celled bacteria.
Ecosystem Sampling
[0645] Metagenomic datasets were acquired from human fecal and oral
samples, fecal samples from other animals, freshwater lakes and
rivers, marine ecosystems, sediments, hot springs, soils, deep
subsurface habitats and the built environment (FIG. 5). For a
subset of these, analyses of bacterial, archaeal and eukaryotic
organisms were published previously. Genome sequences that were
clearly not bacterial, archaeal, archaeal virus, eukaryotic or
eukaryotic virus were classified as either phage or plasmid-like
based on their gene inventories. De novo assembled fragments of
close to or >200 kbp in length were tested for circularization
and a subset selected for manual verification and curation to
completion (see Methods).
Genome Sizes and Basic Features 358 phage, 3 plasmid and 4
phage-plasmid sequences were reconstructed (FIG. 5).
[0646] Additional sequences inferred to be plasmids were excluded
(see Methods), and only those encoding CRISPR-Cas loci were
retained (see below). Consistent with classification as phage, a
wide variety of phage-relevant genes were identified, including
those involved in lysis and encoding structural proteins, and other
expected phage genomic features were documented. Some phage
predicted proteins are large, up to 7694 amino acids in length.
Many of these were tentatively annotated as structural proteins.
180 phage sequences were circularized and 34 were manually curated
to completion, in some cases by resolving complex repeat regions
and their encoded proteins (see Methods). Some genomes show a clear
GC skew signal for bi-directional replication, information that
constrains their replication origin. The three largest complete,
manually curated and circularized phage genomes are 634, 636 and
643 kbp in length and represent the largest phage genomes reported
to date. Previously, the largest circularized phage genome was 596
kbp in length (Paez-Espino et al. (2016) supra). The same study
reported a circularized genome of 630 kbp in length, but this is an
artifact. The problem of concatenated sequences was sufficiently
prominent in IMG-VR that these data were not included in further
analyses. The complete and circularized genomes from the study,
Refseq and published research were used to depict a current view of
the distribution of phage genome sizes (Methods). The median genome
size for complete phage is .about.52 kbp (FIG. 1A), similar to the
average size of .about.54 kbp reported previously (Paez-Espino et
al. (2016) supra). Thus, sequences reported here substantially
expand the inventory of phage with unusually large genomes (FIG.
1B).
[0647] Intriguingly, two related sequences of 712 and >716 kbp
in length were identified and manually curated (FIG. 5). These were
classified as phage based on their overall genome content and the
presence of terminase genes. The assemblies are confounded by few
kb-long complex regions comprised of small repeats at both genome
ends. It is anticipated that these genomes could be closed if the
repeat regions could be rationalized.
[0648] Some genomes have very low coding density (nine <75%) due
to use of a genetic code different from that used for gene
prediction. A similar phenomenon was reported for Lak phage (Devoto
et al. (2019) Nat Microbiol, and Ivanova et al. (2014) Science 344:
909-913). Distinct from prior studies, the genomes appear to use
genetic code 16, in which TAG, normally a stop codon, codes for an
amino acid.
[0649] In only one case, a sequence of >200 kbp that was
classified as a prophage based on transition into flanking
bacterial genome sequence was identified. However, around half the
genomes were not circularized, so their derivation from prophage
cannot be ruled out. The presence of integrases in some genomes is
suggestive of a lysogenic lifestyle under some conditions.
Hosts, Diversity and Distribution
[0650] An intriguing question relates to the evolutionary history
of phage with huge genomes. Are they the result of recent genome
expansion within clades of normal sized phage or is a large
inventory of genes an established, persistent strategy? To
investigate this, phylogenetic trees for the large terminase
subunit (FIG. 2) and major capsid proteins using as context
sequences in public databases for phage of all sizes were
constructed (Methods). Many of the sequences from the large phage
genomes cluster together, defining clades. Analysis of the genome
size information for database sequences shows that the public
sequences that fall into these clades are from phage with genomes
of at least 120 kbp in length. The largest clade, referred to here
as Mahaphage (Maha being Sanskrit for huge), includes all of the
present study's largest genomes as well as the Lak genomes from
human and animal microbiomes (Devoto et al. (2019) supra). Six
other clearly defined clusters of large phage were identified, and
they were named using the word for "huge" in a variety of
languages. The existence of these clades establishes that large
genome size is a relatively stable trait. Within the seven clades,
phage were sampled from a wide variety of environment types,
indicating diversification of these large phage and their hosts
across ecosystems. The environmental distribution of phage that are
closely enough related that their genomes largely can be aligned
was also examined. In 17 cases, these phage occur in at least two
biotope types.
[0651] To determine the extent to which bacterial host phylogeny
correlates with phage clades, phage hosts were identified using
CRISPR spacer targeting from bacteria in the same or related
samples and phylogeny of normally host-associated genes that occur
on phage (see below). The predictive value of bacterial
affiliations of the phage gene inventories was also tested
(Methods) and it was found that in every case, CRISPR spacer
targeting and phylum-level phylogenetic profiling agreed with gene
inventory characterizations. Consequently, this method was used to
predict the phylum-level affiliations of hosts for many phage. The
results establish the importance of firmicute and proteobacterial
hosts, and indicate the higher prevalence of firmicute phage in the
human and animal gut compared to other environments (FIG. 5).
Notably, the four largest genomes (634-716 kbp in length) are all
for phage predicted to replicate in Bacteroidetes, as do Lak phage
with 540-552 kbp genomes (Devoto et al. (2019) supra), and all
cluster within Mahaphage. Overall, phage grouped together
phylogenetically are predicted to replicate in bacteria of the same
phylum.
Metabolism, Transcription, Translation
[0652] The phage genomes encode proteins predicted to localize to
the bacterial membrane or cell surface. These may impact the
susceptibility of the host to infection by other phage. Almost all
previously reported categories of genes suggested to augment host
metabolism during infection were identified. Many phage have genes
involved in steps of de novo biosynthesis of purines and
pyrimidines and multiple steps that interconvert nucleic and
ribonucleic acids and nucleotide phosphorylation states. These gene
sets are intriguingly similar to those of bacteria with very small
cells and putative symbiotic lifestyles (Castelle and Banfield
(2018) Cell 172: 1181-1197).
[0653] Notably, many phage have genes whose predicted functions are
in transcription and translation. Phage encode up to 64 tRNAs per
genome, with sequences distinct from those of their hosts.
Generally, the number of tRNAs per genome increases with genome
length (FIG. 1). They often have up to 16 tRNA synthetases per
genome, that are related to, but distinct from, those of their
hosts. Phage may use these proteins to charge their own tRNA
variants with host-derived amino acids. A subset of genomes have
genes for tRNA modification and to repair tRNAs cleaved as part of
host defense against phage infection. Also identified are up to
three probable ribosomal proteins per genome, the most common of
which is rpS21 (a phenomenon only recently reported in phage)
(Mizuno et al. (2019) Nat. Commun. 10: 752); FIG. 3). Intriguingly,
it is noted that the phage rpS21 sequences have N-terminal
extensions rich in arginine, lysine, and phenylalanine: residues
that bind nucleic acids. It is predicted that these phage ribosomal
proteins substitute for host proteins in the ribosome (Mizuno et
al. (2019) supra), and that the extensions protrude from the
ribosome surface near the site of translation initiation to
localize the phage mRNAs.
[0654] Some phage have genes predicted to function in other protein
synthesis steps, including to ensure efficient translation. Several
encode either initiation factor 1 or 3 or both, sometimes as well
as elongation factors G, Tu, Ts and release factors. Also
identified are genes that encode ribosome recycling factors, along
with tmRNAs and small protein B (SmpB) that rescue ribosomes
stalled on damaged transcripts and trigger the degradation of
aberrant proteins. tmRNAs are also used by phages to sense the
physiological state of host cells and can induce lysis when the
number of stalled ribosomes in the host is high.
[0655] These observations suggest many ways in which some large
phage can substantially intercept and redirect ribosome function.
As phage mRNA sequences need to engage with the 3' end of the host
16S rRNA to initiate translation, their mRNA ribosomal binding
sites were predicted. In the majority of cases, phage mRNAs have
canonical Shine Dalgarno (SD) sequences, and an additional
.about.15% have non-standard SD binding sites. Interestingly,
however, phage whose genomes encode a probable or possible rpS1
rarely have identifiable or canonical SD sequences. Thus,
phage-encoded rpS1 may selectively initiate translation of phage
mRNAs. Overall, phage genes appear to redirect the host's protein
production capacity to favor phage genes by intercepting the
earliest steps of translation. These inferences are aligned with
findings for some eukaryotic viruses, which control every phase of
protein synthesis (Jaafar and Kieft (2019) Nat. Rev. Microbiol.
17:110-123). Interestingly, some large putative plasmids also have
analogous suites of translation relevant genes.
[0656] About half of the phage genomes have one to fifty sequences
>25 nt in length that fold into perfect hairpins. The
palindromes (sequences with dyad symmetry) are almost exclusively
intergenic and each is unique within a genome. Some, but not all,
are predicted to be rho-independent terminators, thus provide clues
regarding genes that function as independently regulated units
(Methods). However, some palindromes are up to 74 bp in length, and
34 genomes have examples of >40 nt in length, seemingly larger
than normal terminators. These occur almost exclusively in
Mahaphage and may have alternative or additional functions, such as
modulation of the movement of the mRNA through the ribosome.
CRISPR-Cas Mediated Interactions
[0657] Almost all major types of CRISPR-Cas systems on phage,
including Cas9, the recently described Type V-I (Yan et al. (2019)
Science 363: 88-91), and new subtypes of Type V-F systems were
identified (Harrington et al. (2018) Science 362: 839-842.). The
Class II systems (types II and V) are reported in phage for the
first time. Most effector nucleases (for interference) have
conserved catalytic residues, implying that they may be
functional.
[0658] Unlike the previously well described case of a phage with a
CRISPR system (Seed et al. (2013) Nature 494: 489-491), almost all
phage CRISPR systems lack spacer acquisition machinery (Cas1, Cas2,
and Cas4) and many lack recognizable genes for interference. For
example, two related phage have both a Type I-C variant system
lacking Cas1 and Cas2 and a helicase protein in lieu of Cas3. They
also harbor a second system containing a new candidate .about.750
aa Type V effector protein that occurs proximal to CRISPR arrays.
In some cases, phage lacking genes for interference and spacer
integration have similar CRISPR repeats as their hosts, thus may
use Cas proteins synthesized by their host for these functions.
Alternatively the systems lacking an effector nuclease may repress
transcription of the target sequences without cleavage (Luo et al.
(2015) Nucleic Acids Res. 43:674-681; Stachler and Marchfelder
(2016) J. Biol. Chem. 291:15226-15242).
[0659] The phage-encoded CRISPR arrays are often compact (3-55
repeats; median 6 per array. This range is substantially smaller
than typically found in bacterial genomes (Toms and Barrangou
(2017) Biol. Direct 12:20). Some phage spacers target core
structural and regulatory genes of other phage. Thus, phage
apparently augment their hosts' immune arsenal to prevent infection
by competing phage.
[0660] Several large plasmid or plasmid-like genomes that encode a
variety of types of CRISPR-Cas systems were identified. Some of
these systems also lack Cas1 and Cas2. Most commonly, the spacers
target the mobilization and conjugation-related genes of other
plasmids, as well as nucleases and structural proteins of
phage.
[0661] Some phage-encoded CRISPR loci have spacers that target
bacteria in the same sample or in a sample from the same study. It
is supposed that the targeted bacteria are the hosts for these
phage, an inference supported by other host prediction analyses.
Some loci with bacterial chromosome-targeting spacers encode Cas
proteins that could cleave the host chromosome, and some do not.
Targeting of host genes could disable or alter their regulation,
which may be advantageous during the phage infection cycle. Some
phage CRISPR spacers target bacterial intergenic regions, possibly
interfering with genome regulation by blocking promoters or
silencing non-coding RNAs.
[0662] Among the most interesting examples of CRISPR targeting of
bacterial chromosomes are genes involved in transcription and
translation. For instance, one phage targets a .sigma..sup.70
transcription factor in its host's genome, while encoding the gene
for .sigma..sup.70. There are previous reports of .sigma..sup.70
hijacking by phage with anti-sigma factors This may also occur with
some huge phage whose genomes encode anti-sigma factors. In another
example, a phage spacer targets the host Glycyl tRNA
synthetase.
[0663] Interestingly, no evidence was found of targeting of any
CRISPR-bearing phage by a host-encoded spacer, hinting at yet to be
revealed components in phage-host-CRISPR interactions. However,
phage CRISPR targeting of other phage that are also targeted by
bacterial CRISPR (FOG/4) suggested phage-host associations that
were broadly confirmed by the phage phylogenetic profile.
[0664] Some large Pseudomonas phage encode Anti-CRISPRs (Acr)
(Bondy-Denomy et al. (2015) Nature 526:136-139; Pawluk et al.
(2016) Nat Microbiol 1: 16085) and proteins that assemble a
nucleus-like compartment segregating their replicating genomes from
host defense and other bacterial systems. Proteins encoded in huge
phage genomes that cluster with AcrVA5, AcrVA2, and AcrIIA7 that
may function as Acrs were identified. Also identified were
tubulin-homologs (PhuZ) that position the "phage nucleus", and
proteins related to components of the proteinaceous barrier. Thus,
phage `nuclei` may be a relatively common feature in large
phage.
Methods
Phage and Plasmid Genome Identification
[0665] Datasets generated in the current study, those from prior
research, the Tara Oceans microbiomes (Karsenti et al. (2011) PLoS
Biol. 9:e1001177), and the Global Oceans Virome (GOV; (Roux et al.
(2016) Nature 537:689-693) were searched for sequence assemblies
that could have derived from phage with genomes of >200 kbp in
length. Read assembly, gene prediction, and initial gene annotation
followed standard methods reported previously (Wrighton et al.
(2014) ISME J. 8:1452-1463).
[0666] Phage candidates were initially found by retrieving
sequences that were not assigned to a genome and had no clear
taxonomic profile at the domain level. Taxonomic profiles were
determined through a voting scheme, where there had to be a winner
taxonomy >50% votes at each taxonomic rank based on Uniprot and
ggKbase (ggkbase.berkeley.edu) database annotations. Phages were
further narrowed down by identifying sequences with a high number
of hypothetical protein annotations and/or the presence of phage
structural genes, e.g. capsid, tail, holin. All candidate phage
sequences were checked throughout to distinguish putative prophage
from phage. Prophage were identified based on a clear transition
into genome with a high fraction of confident functional
predictions, often associated with core metabolic functions, and
much higher similarity to bacterial genomes. Plasmids were
distinguished from phage based on matches to plasmid marker genes
(e.g. parA). Three sequence assemblies could not unambiguously be
distinguished between phage and plasmid, and were assigned as
"phage-plasmid".
Phage and Plasmid Genome Manual Curation
[0667] All scaffolds classified as phage or phage-like were tested
for end overlaps using a custom script and checked manually for
overlap. Assembled sequences that could be perfectly circularized
were considered potentially "complete". Erroneous concatenated
sequence assemblies were initially flagged by searching for direct
repeats >5 kb using Vmatch (Kurtz (2003) Ref Type: Computer
Program 412:297). Potentially concatenated sequence assemblies were
manually checked for multiple large repeating sequences using the
dotplot and RepeatFinder features in Geneious v9. Sequences were
corrected and removed from further analysis if the corrected length
was <200 kbp.
[0668] A subset of the phage sequences was selected for manual
curation, with the goal of finishing (replacing all N's at
scaffolding gaps or local misassemblies by the correct nucleotide
sequences and circularization). Curation generally followed methods
described previously (Devoto et al. (2019) supra). In brief, reads
from the appropriate dataset were mapped using Bowtie2 (Langmead
and Salzberg (2012) Nat. Methods 9:357-359) to the de novo
assembled sequences. Unplaced mate pairs of mapped reads were
retained with shrinksam (github.com/bcthomas/shrinksam). Mappings
were manually checked throughout to identify local misassemblies
using Geneious v9. N-filled gaps or misassembly corrections made
use of unplaced paired reads, in some cases using reads relocated
from sites where they were mis-mapped. In such cases, mis-mappings
were identified based on much larger than expected paired read
distances, high polymorphism densities, backwards mapping of one
read pair, or any combination of the aforementioned.
[0669] Similarly, ends were extended using unplaced or incorrectly
placed paired reads until circularization could be established. In
some cases, extended ends were used to recruit new scaffolds that
were then added to the assembly. The accuracy of all extensions and
local assembly changes were verified in a subsequent phase of read
mapping. In many cases, assemblies were terminated or internally
corrupted by the presence of repeated sequences. In these cases,
blocks of repeated sequence as well as unique flanking sequence
were identified. Reads were then manually relocated, respecting
paired read placement rules and unique flanking sequences. After
gap closure, circularization, and verification of accuracy
throughout, end overlap was eliminated, genes were predicted and
throughout, and the start moved to an intergenic region, in some
cases suspected to be origin based on a combination of coverage
trends and GC skew (Brown et al. (2016) Nat. Biotechnol.
34:1256-1263). Finally, the sequences were checked to identify any
repeated sequences that could have led to an incorrect path choice
because the repeated regions were larger than the distance spanned
by paired reads. This step also ruled out artifactual long phage
sequences generated by end to end repeats of smaller phage, which
occur in previously described datasets.
Structural and Functional Annotation
[0670] Following identification and curation of phage genomes,
coding sequences (CDS) were predicted with prodigal (-m -c -g 11 -p
single) with genetic code 11. The resulting CDS were annotated as
previously described by searching against UniProt, UniRef, and KEGG
(Wrighton et al. (2014) supra). Functional annotations were further
assigned by searching proteins against Pfam r32 (Finn et al. (2014)
Nucleic Acids Res. 42:D222-30), TIGRFAMS r15 (Haft et al. (2013)
Nucleic Acids Res. 41:D387-95), and Virus Orthologous Groups r90
(vogdb.org). tRNAs were identified with tRNAscan-SE 2.0 (Lowe and
Eddy, (1997) Nucleic Acids Res. 25: 955-964) using the bacterial
model. tmRNAs were assigned using ARAGORN v1.2.38 (Laslett and
Canback, (2004) Nucleic Acids Res. 32: 11-16) with the
bacterial/plant genetic code. Clustering of the protein sequences
into families was achieved using a two-step procedure. A first
protein clustering was done using the fast and sensitive protein
sequence searching software MMseqs (Hauser et al. (2016)
Bioinformatics 32: 1323-1330). An all-vs-all sequences search was
performed using e-value: 0.001, sensitivity: 7.5 and coverage: 0.5.
A sequence similarity network was built based on the pairwise
similarities and the greedy set cover algorithm from MMseqs was
performed to define protein subclusters. The resulting subclusters
were defined as subfamilies. In order to test for distant homology,
subfamilies were grouped into protein families using an HMM-HMM
comparison. The proteins of each subfamily with at least two
protein members were aligned using the result2msa parameter of
mmseqs2, and from the multiple sequence alignments HMM profiles
were built using the HHpred suite. The subfamilies were then
compared to each other using HHblits (Remmert et al. (2011) Nat.
Methods 9: 173-175 from the HHpred suite (with parameters -v 0 -p
50 -z 4 -Z 32000 -B 0 -b 0). For subfamilies with probability
scores of .gtoreq.95% and coverage .gtoreq.0.50, a similarity score
(probability X coverage) was used as weights of the input network
in the final clustering using the Markov Clustering algorithm, with
2.0 as the inflation parameter. These clusters were defined as the
protein families Hairpins (palindromes, based on identical
overlapping repeats in the forward and reverse directions) were
identified using the Geneious Repeat Finder and located
dataset-wide using Vmatch (Kurtz (2003) supra). Repeats >25 bp
with 100% similarity were tabulated.
Reference Genomes for Size Comparisons
[0671] RefSeq v92 genomes were recovered by using the NCBI Virus
portal and selecting only complete dsDNA genomes with bacterial
hosts. Genomes from (Paez-Espino et al. (2016) supra) were
downloaded from IMG/VR and only sequence assemblies labeled
"circular" with predicted bacterial hosts were retained. Many of
the genomes were the result of erroneous concatenated repeating
assemblies. Given the presence of sequences in IMG/VR that are
based on erroneous concatenations, the study only considered
sequences from this source that are >200 kb; a subset of these
were removed as artifactual sequences.
Host Prediction
[0672] The phylum affiliations of bacterial hosts for phage were
predicted by considering the Uniprot taxonomic profiles of every
CDS for each phage genome. The phylum level matches for each phage
genome were summed and the phylum with the most hits was considered
as the potential host phylum. However, only cases where this phylum
that had 3.times. as many counts as the next most counted phylum
were assigned as the tentative phage host phylum. Phage hosts were
further assigned and verified using CRISPR targeting. CRISPR arrays
were predicted on sequence assemblies >1 kbp from the same
environment that each phage genome was reconstructed. Spacers were
extracted and searched against the genomes from the same site using
BLASTN -short (Altschul et al. (1990) J. Mol. Biol. 215:403-410).
Sequence assemblies containing spacers with a match of length
>24 bp and .ltoreq.1 mismatch or at least 90% sequence identity
to a genome were considered targets. In the case of phage, the
match was used to infer a phage-host relationship. In all cases,
the predicted host phylum based on taxonomic profiling and CRISPR
targeting were in complete agreement. Similarly, the phyla of hosts
were predicted based on phylogenetic analysis of phage genes also
found in host genomes (e.g., involved in translation and nucleotide
reactions). Inferences based on computed taxonomic profiles and
phylogenetic trees were also in complete agreement.
Alternative Genetic Codes
[0673] In cases where gene prediction using the standard bacterial
code (code 11) resulted in seemingly anomalously low coding
densities, potential alternative genetic codes were investigated.
In addition to making a prediction using Fast and Accurate genetic
Code Inference and Logo (FACIL; (Dutilh et al. (2011)
Bioinformatics 27:1929-1933)), genes with well defined functions
(e.g., polymerase, nuclease) were identified and the stop codons
terminating genes that were shorter than expected were determined.
Genes were then re-predicted using Glimmer and Prodigal set such
that codon was not interpreted as a stop. Other combinations of
repurposed stop codons were evaluated, and candidate codes (e.g.,
code 6, with only one stop codon) were ruled out due to unlikely
gene fusion predictions.
[0674] Introns were identified in some longer than expected
pseudo-tRNAs by re-predicting the tRNAs using eukaryotic settings
(as tRNA scan does not expect introns in tRNA genes in bacteria and
phage).
Terminase Phylogenetic Analysis
[0675] The large terminase phylogenetic tree was constructed by
recovering large terminases from the aforementioned annotation
pipeline. CDS that matched with >30 bitscore against PFAM,
TIGRFAMS, and VOG were retained. Any CDS that had a hit to large
terminase, regardless of bitscore, was searched using HHblits
(Steinegger et al. Bioinformatics 21:951-960) against the
uniclust30_2018_08 database. The resulting alignment was then
further searched against the PDB70 database. Remaining CDS that
clustered in protein families with a large terminase HMM were also
included after manual verification. Detected large terminases were
manually verified using HHPred (Steinegger et al. supra) and jPred
(Cole et al. (2008) Nucleic Acids Res. 36:W197-201). Large
terminases from the >200 kb (Paez-Espino et al. (2016) supra)
phage genomes and all >200 kb complete dsDNA phage genomes from
RefSeq r92 were also included by protein family clustering with the
phage CDS from this study. The resulting terminases were clustered
at 95% amino acid identity (AAI) to reduce redundancy using cd-hit
(Huang et al. (2010) Bioinformatics 26:680-682). Smaller phage
genomes were included by searching the resulting CDS set against
the Refseq protein database and retaining the top 10 best hits.
Those hits that had no large terminase match against PFAM,
TIGRFAMS, or VOG were removed from further consideration and the
remaining set was clustered 90% AAI. The final set of large
terminase CDS were aligned MAFFT v7.407 (--localpair --maxiterate
1000) and poorly aligned sequences were removed and the resulting
set was realigned. The phylogenetic tree was inferred using IQTREE
v1.6.9 (Nguyen et al. (2015) Mol. Biol. Evol. 32:268-274).
Phage Encoded tRNA Synthetase Trees
[0676] Phylogenetic trees were constructed for phage encoded tRNA
synthetase, ribosomal and initiation factor protein sequences using
a set of the closest set of reference from NCBI and bacterial
genomes from the current study.
CRISPR-Cas Locus Detection and Host Identification
[0677] Phage-encoded CRISPR-Cas loci were identified using the same
methods as used to identify bacterial CRISPR-Cas loci, spacers
extracted from between repeats of the CRISPR locus using MinCED
(github.com/ctSkennerton/minced) and CRISPRDetect (Biswas et al.,
2016) were compared to sequences reconstructed from the same site
and targets classified as bacterial, phage or other.
[0678] Because many phage hosts cannot be identified by CRISPR
targeting (perhaps because phage had proliferated in samples
containing sensitive hosts, or the targets are sufficiently mutated
to avoid spacer detection) additional lines of evidence were used
to propose host identities. Due to uncertainty in these methods,
possible phage predictions were made only at the phylum level. In
this analysis, the fraction of genes encoded on any genome with the
best predicted protein match to each phylum was computed. Only in
cases where the most highly represented phylum exceeded in
frequency the next most common phylum by .gtoreq.3.times. was a
tentative bacterial host proposed. This threshold was verified as
conservative, based on confirmed host phylum information from
CRISPR targeting or phylogenetic analysis.
Data Availability
[0679] Supplementary document "Genbank" includes the Genbank format
files for the genome sequences reported in this study. All reads
are being deposited in the short read archive (if not already
lodged there) and genome sequences in NCBI.
Example 2
[0680] Cas12J represents the smallest known single-effector Cas
protein with double-stranded DNA (dsDNA) targeting ability. Cas12J
is capable of cleaving dsDNA without a requirement for an accessory
RNA (e.g. such as a tracrRNA) to function. Additionally, the RuvC
domain, which is the a highly conserved domain across Cas12 and
Cas9, is highly divergent in Cas12J from known Cas proteins, and
the domain architecture is different across members of the Cas12
protein superfamily.
Results
[0681] To investigate the functionality and DNA targeting
capability of the Cas12J effector in a heterologous context, an
efficiency of transformation (EOT) plasmid interference assay was
set up (FIG. 11A). Escherichia coli BL21(DE3) expressing cas12J and
a crRNA guide targeting the antisense strand of the bla gene, or a
non-targeting guide, were transformed with pUC19 (FIG. 11B). The
assay revealed that the pUC19 transformation efficiency is reduced
by 2-3 orders of magnitude in strains producing Cas12J and the
pUC19 targeting guide, compared to strains producing Cas12J and the
non-targeting guide (FIG. 11C). This result is indicative of a
robust and guide dependent double-stranded DNA interference
activity of Cas12J. To assess the DNA interference unbiased
relative transformation efficiency of each strain, the pYTK001
plasmid was transformed as a control (FIG. 11B). The transformation
efficiency revealed that the strains are equally competent for
transformation of a non-targeted plasmid (FIG. 11C).
Methods
Cloning of the Expression Plasmids
[0682] The gene sequence of cas12J from contig
P0_An_GD2017L_S7_coassembly_k141_3339380 was ordered as a G-block
from IDT and cloned into pRSFDuet-1 (Novagen) into MCSI using
Golden Gate assembly. In the same reaction a T7 promotor, the
respective consensus repeat sequence from the CRISPR-array located
on contig P0_An_GD2017L_S7_coassembly_k141_3339380, together with a
35 bp spacer amenable to Golden Gate assembly mediated spacer
exchange were introduced downstream of the cas12J ORF in place of
MCSII. In the same reaction a hepatitis delta virus ribozyme
(HDVrz) was introduced downstream of the spacer to facilitate
homogeneous processing of the immature crRNA transcript at its
3'-terminus. To generate the pUC19 targeting Cas12J-vector, the
non-targeting spacer was exchanged by Golden Gate assembly to a
sequence matching base pairs 11-45 of the pUC19 bla gene downstream
of the AGTATTC sequence, to allow for production of an antisense
strand complementary crRNA guide.
Plasmid Interference Assay
[0683] The generated Cas12J vectors (non-targeting and
pUC19-targeting) were transformed in chemically competent E. coli
BL21(DE3) (NEB). Three individual colonies for each strain (A, B
and C strains) were picked to inoculate three 5 mL (LB, Kanamycin
50 .mu.g/mL) starter cultures to prepare electrocompetent cells the
following day. 50 mL (LB, Kanamycin 50 .mu.g/mL) main cultures were
inoculated 1:100 and grown vigorously shaking at 37.degree. C. to
an OD.sub.600 of 0.3. Subsequently, the cultures were cooled to
room temperature and cas12J expression was induced with 0.2 mM
IPTG. Cultures were grown to an OD.sub.600 of 0.6-0.7 at 25.degree.
C. for 1 h, before preparation of electrocompetent cells by
repeated ice-cold ddH.sub.20 and 10% glycerol washes. Cells were
resuspended in 250 .mu.L 10% glycerol. 90 .mu.L aliquots were flash
frozen in liquid nitrogen and stored at -80.degree. C. The next
day, 80 .mu.L competent cells were combined with 3.2 .mu.L plasmid
(20 ng/.mu.L pUC19 target plasmid, or 20 ng/.mu.L pYTK001 control
plasmid), incubated for 30 min on ice and split into three
individual 25 .mu.L transformation reactions. After electroporation
in 0.1 mm electroporation cuvettes (Bio-Rad) on a Micropulser
electroporator (Bio-Rad), cells were recovered in 1 mL recovery
medium (Lucigen) supplemented with 0.2 mM IPTG, shaking at
37.degree. C. for one hour. Subsequently, 10-fold dilution series
were prepared and 5 of the respective dilution steps were
spot-plated on LB-Agar containing the appropriate antibiotics.
Plates were incubated over night at 37.degree. C. and colonies were
counted the following day to determine the transformation
efficiency. To assess the transformation efficiency, the mean and
standard deviations were calculated from the cell forming units per
ng transformed plasmids for the electroporation triplicates.
[0684] FIG. 11A-11C shows the efficiency of transformation plasmid
interference assay. FIG. 11A upper panel: experimental scheme. E.
coli producing Cas12J are transformed with a targeted plasmid
(pUC19). Lower panel: vector map of the effector expression
plasmid. FIG. 11B, serial dilutions of E. coli producing Cas12J and
either pUC19-targeting or non-targeting guides, transformed with
pUC19 (left) or pYTK001 (right). FIG. 11C, calculated
transformation efficiencies in cell forming units (cfu) per ng
transformed plasmid. Mean and +/-s.d. (error bars) values were
derived from triplicates.
Example 3
Results
[0685] To demonstrate that Cas12J cuts dsDNA--in vitro experiments
outside of cells (i.e., in a non-cellular context) were performed.
Linear dsDNA was cleaved in the presence of Cas12J and a guide RNA
designed to hybridize to a target sequence adjacent to a PAM motif.
The Cas12J ribonucleoprotein (RNP) complex was either assembled
inside of cells (E. coli in this case via the introduction of
plasmid DNA encoding the protein and the guide RNA), or assembled
in vitro outside of cells from apo protein and synthetic RNA
oligonucleotides. The experiment revealed that RNPs with
Cas12J-1947455 ("Ortholog #1"), Cas12J-2071242 ("Ortholog #2"), or
Cas12J-3339380 ("Ortholog #3") assembled either inside or outside
of cells cleaved linear dsDNA fragments guided by the crRNA spacer
sequence of the guide RNA (FIG. 12A and FIG. 12B). The 1.9 kb
linear DNA substrate was cleaved into 1.2 kb and a 0.7 kb fragment,
indicative of an endonucleolytic DNA double strand cleavage event
close to the site of guide complementarity. dsDNA cleavage was not
observed in the absence of a guide complementary site on the DNA.
This experiment demonstrated that Cas12J (e.g., Cas12J-1947455,
Cas12J-2071242 and Cas12J-3339380) is a crRNA guided
DNA-endonucleases capable of introducing double strand breaks into
DNA. Furthermore, the experiment demonstrated that functional
Cas12J RNPs can be assembled inside and/or outside of cells.
[0686] FIG. 12A-12B demonstrates that Cas12J (e.g., Cas12J-1947455,
Cas12J-2071242 and Cas12J-3339380) cleave linear dsDNA fragments
guided by a crRNA spacer sequence. FIG. 12A, Time dependent dsDNA
cleavage assays for the RNPs that were assembled inside of cells.
top: Cas12J-1947455 (Cas12J-1), middle: Cas12J-2071242 (Cas12J-2)
and bottom: Cas12J-3339380 (Cas12J-3). The far right lanes are
non-complementary DNA controls, which could not be identified by
the respective crRNA guide. FIG. 12B, Time dependent dsDNA cleavage
assays for the RNPs that were assembled in vitro outside of cells.
top: Cas12J-1947455 (Cas12J-1), middle: Cas12J-2071242 (Cas12J-2)
and bottom: Cas12J-3339380 (Cas12J-3). The far right lanes are
non-complementary DNA controls, which could not be identified by
the respective crRNA guide.
[0687] PAM depletion assays were performed in Escherichia coli. In
the assay, Cas12J targets a DNA sequence adjacent to a randomized
sequence in a plasmid library. NGS sequencing revealed that Cas12J
and crRNA were sufficient in bacteria to deplete plasmids with
crRNA guide complementary target DNA sites, when a T-rich PAM
sequence was adjacent to the protospacer (FIG. 13). The experiment
also showed that no tracrRNA was required for the formation of
functional effectors. Noteworthy, ortholog #2 features a minimal
5'-TBN-3' PAM sequence.
[0688] FIG. 13. PAM sequences depleted by the three different
orthologs, demonstrating that PAMs are straightforward to identify
for any desired Cas12J protein.
Methods
Cloning of the Expression Constructs
[0689] The gene sequences of Cas12J-1947455, Cas12J-2071242 and
Cas12J-3339380 were ordered as G-blocks from IDT and cloned into
pRSFDuet-1 (Novagen) into MCSI C-terminally fused to a
hexa-histidine tags using Golden Gate assembly. For co-expression
of cas12J with crRNA guides, CRISPR-arrays (36 bp repeat followed
by a 35 bp spacer, six units thereof) were cloned under the control
of a T7-promoter in high copy vectors (ColE1 origin), which
contained bla genes for selection.
Production of the Cas12J-RNP In Vivo and Purification
[0690] The generated cas12J overexpression vectors and CRISPR array
expression vectors were co-transformed in E. coli BLR(DE3)
(Novagen) and incubated over night at 37.degree. C. on LB-Kan-Carb
agar plates (50 .mu.g/mL Kanamycin, 50 .mu.g/mL Carbenicillin).).
Single colonies were picked to inoculate 80 mL (LB, Carbenicillin
50 .mu.g/mL and Kanamycin 50 .mu.g/mL) starter cultures which were
incubated at 37.degree. C. shaking vigorously overnight. The next
day, 1.5 L TB-Kan-Carb medium (Carbenicillin 50 .mu.g/mL and
Kanamycin 50 .mu.g/mL) were inoculated with the respective 40 mL
starter culture and grown at 37.degree. C. to an OD.sub.600 of 0.6,
cooled down on ice for 15 min and gene expression was subsequently
induced with 0.5 mM IPTG followed by incubation over night at
16.degree. C. Cells were harvested by centrifugation and
resuspended in wash buffer (50 mM HEPES-Na (pH 7.5), 500 mM NaCl,
20 mM imidazole, 5% glycerol and 0.5 mM TCEP), subsequently lysed
by sonication followed by lysate clarification by centrifugation.
The soluble fraction was loaded on a 5 mL Ni-NTA Superflow
Cartridge (Qiagen) pre-equilibrated in wash buffer. Bound proteins
were washed with 20 column volumes (CV) wash buffer and
subsequently eluted in 3 CV elution buffer (50 mM HEPES-Na (pH
7.5), 500 mM NaCl, 500 mM imidazole, 5% glycerol and 0.5 mM TCEP).
Eluted proteins were dialyzed over night at 4.degree. C. in
slide-a-lyzer dialysis cassettes 10k mwco (Thermo Fisher
Scientific) against ion-exchange (IEX) loading buffer (20 mM Tris
pH 9.0, 4.degree. C., 125 mM NaCl, 5% glycerol and 0.5 mM TCEP).
Proteins were loaded onto 2.times.5 mL HiTrap Q HP anion exchange
chromatography columns. Proteins were eluted in a gradient of IEX
elution buffer (20 mM Tris pH 9.0, 4.degree. C., 1 M NaCl, 5%
glycerol and 0.5 mM TCEP). Elution fractions were analyzed by
SDS-PAGE and Urea-PAGE and fraction containing RNP formed by Cas12J
and crRNA were concentrated to 1 mL. Finally, proteins were
injection into a HiLoad 16/600 Superdex 200 pg column
pre-equilibrated in size-exclusion buffer (10 mM HEPES-Na (pH 7.5),
150 mM NaCl and 0.5 mM TCEP). Peak fractions were concentrated to
an absorption at 280 nm of 60 AU (NanoDrop 8000 Spectrophotometer,
Thermo Scientific), corresponding to an estimated concentration of
500 .mu.M. Subsequently, proteins were snap frozen in liquid
nitrogen and stored at -80.degree. C.
Production and Purification of Apo Cas12J
[0691] The generated cas12J overexpression vectors were transformed
in chemically competent E. coli BL21(DE3) (NEB) and incubated over
night at 37.degree. C. on LB-Kan agar plates (50 .mu.g/mL
Kanamycin). Single colonies were picked to inoculate 80 mL (LB,
Kanamycin 50 .mu.g/mL) starter cultures which were incubated at
37.degree. C. shaking vigorously overnight. The next day, 1.5 L
TB-Kan medium (50 .mu.g/mL Kanamycin) were inoculated with the
respective 40 mL starter culture and grown at 37.degree. C. to an
OD.sub.600 of 0.6, cooled down on ice for 15 min and gene
expression was subsequently induced with 0.5 mM IPTG followed by
incubation over night at 16.degree. C. Cells were harvested by
centrifugation and resuspended in wash buffer (50 mM HEPES-Na (pH
7.5), 1 M NaCl, 20 mM imidazole, 5% glycerol and 0.5 mM TCEP),
subsequently lysed by sonication followed by lysate clarification
by centrifugation. The soluble fraction was loaded on a 5 mL Ni-NTA
Superflow Cartridge (Qiagen) pre-equilibrated in wash buffer. Bound
proteins were washed with 20 column volumes (CV) wash buffer and
subsequently eluted in 5 CV elution buffer (50 mM HEPES-Na (pH
7.5), 500 mM NaCl, 500 mM imidazole, 5% glycerol and 0.5 mM TCEP).
The eluted proteins were concentrated to 1 mL before injection into
a HiLoad 16/600 Superdex 200 pg column pre-equilibrated in
size-exclusion buffer (20 mM HEPES-Na (pH 7.5), 500 mM NaCl, 5%
glycerol and 0.5 mM TCEP). Peak fractions were concentrated to an
absorption at 280 nm of 40 AU (NanoDrop 8000 Spectrophotometer,
Thermo Scientific), corresponding to an estimated concentration of
500 .mu.M. Subsequently, proteins were snap frozen in liquid
nitrogen and stored at -80.degree. C.
Cas12J-crRNA RNP Reconstitution
[0692] Cas12J-crRNA RNP complexes were assembled at a concentration
of 1.25 .mu.M by mixing protein and synthetic crRNA (IDT) in a 1:1
molar ratio in reconstitution buffer (10 mM Hepes-K pH 7.5, 150 mM
KCl, 5 mM MgCl.sub.2, 0.5 mM TCEP) and incubation at 20.degree. C.
for 30 min. The synthetic crRNA was prior to the assembly reaction
heated to 95.degree. C. for 3 min and then cooled down to RT for
proper folding.
DNA Cleavage Assay
[0693] DNA target substrates were generated by PCR from plasmid
template DNA. Cleavage reactions were initiated by addition of DNA
(10 nM) to preformed RNP (1 .mu.M) in reaction buffer (10 mM
Hepes-K pH 7.5, 150 mM KCl, 5 mM MgCl.sub.2, 0.5 mM TCEP). The
reactions were incubated at 37.degree. C. and aliquots were removed
at the indicated intervals, quenched with 50 mM EDTA and stored in
liquid nitrogen. After completion of the time-series, samples were
thawed and treated with 0.8 units proteinase K (NEB) for 20 min at
37.degree. C. Loading dye was added (Gel Loading Dye Purple 6X,
NEB) and samples were analyzed by electrophoresis on an 1% agarose
gel.
TABLE-US-00015 Sequences Used crRNA guides: >crRNA-1 (guide
sequence/targeting sequence is in bold)
CACAGGAGAGAUCUCAAACGAUUGCUCGAUUAGUCGAGACAGCUGGUAAUGGGA UACCUU (SEQ
ID NO: 99) >crRNA-2 (guide sequence/targeting sequence is in
bold) UAAUGUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGACUGCCGCCUCCGCGA CGCCCA
(SEQ ID NO: 100) >crRNA-3 (guide sequence/targeting sequence is
in bold) AUUAACCAAAACGACUAUUGAUUGCCCAGUACGCUGGGACUAUGAGCUUAUGUA
CAUCAA (SEQ ID NO: 101) DNA targets (PAM motifs are underlined
crRNA spacer complementary sequences are bold): >Linear
pTarget1:
gctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaa-
cgacttcggg
gcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatctt-
cagcatcttttactttcacca
gcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttga-
atactcatactatcct
tatcaatattattgaagcatttatcagggttattgtacatgagcggatacatatttgaatgtatttagaaaaat-
aaacaaataggggaccgcgca
cataccccgaaaagtgccacctgtcatgaccaaaatcccttaacgtgagttacgttccactgagcgtcagaccc-
cgtagaaaagatcaaag
gatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtg-
gtttgtttgccggatcaagag
ctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagcc-
gtagttaggccaccacttcaa
gaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagt-
cgtgtcttaccgggttggac
tcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggacgtgcacacagcccagcttggag-
cgaacgacctaca
ccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtat-
ccggtaagcggcag
ggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatattatagtcctgtcgggatcgc-
cacctctgacttgag
cgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggtt-
cctggccttttgctggcctt
ttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtgcggccgccccttgtaGTTAagct-
ggtaatgggataccttAt
acagcggccgcgattatcaaaaaggatcacacctagatccttttaaattaaaaatgaagttttaaatcaatcta-
aagtatatatgagtaaacttg
gtctgacagttaccaatgataatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgc-
ctgactccccgtcgtgtagat
aactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgggacccacgctcaccggctc-
cagatttatcagcaata
aaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattg-
ttgccgggaagctagag
taagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcg-
tttggtatggcttcattcagct
ccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcct-
ccgatcgttgtcagaagta
agttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaaga-
tgcttttctgtgactggtgagta ctcaaccaagtcattctgagaatagtgtatgcggcg (SEQ
ID NO: 102) >linear pTarget2:
gctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaa-
cgacttcggg
gcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatctt-
cagcatcttttactttcacca
gcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttga-
atactcatactcttcct
ttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaa-
ataaacaaataggggttccgcgca
catttccccgaaaagtgccacctgtcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagac-
cccgtagaaaagatcaaag
gatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtg-
gtttgtttgccggatcaagag
ctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagcc-
gtagttaggccaccacttcaa
gaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagt-
cgtgtcttaccgggttggac
tcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttgga-
gcgaacgacctaca
ccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtat-
ccggtaagcggcag
ggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttc-
gccacctctgacttgag
cgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggtt-
cctggccttttgctggcctt
ttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtgcggccgccccttgtatTTCTGCC-
GCCTCCGCGA
CGCCCAatacagcggccgcgattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaa-
atcaatctaaagtatata
tgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgtt-
catccatagttgcctgactcccc
gtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgggacccacg-
ctcaccggctccagatt
tatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccag-
tctattaattgttgccgg
gaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtc-
acgctcgtcgtttggtatggc
ttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagct-
ccttcggtcctccgatcgtt
gtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgcc-
atccgtaagatgcttttctgtg
actggtgagtactcaaccaagtcattctgagaatagtgtatgcggcg (SEQ ID NO: 103)
>linear pTarget3:
gctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaa-
cgttcttcggg
gcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatctt-
cagcatcttttactttcacca
gcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttga-
atactcatactcttcct
ttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaa-
ataaacaaataggggttccgcgca
catttccccgaaaagtgccacctgtcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagac-
cccgtagaaaagatcaaag
gatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtg-
gtttgtttgccggatcaagag
ctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgacttctagtgtagccg-
tagttaggccaccacttcaa
gaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagt-
cgtgtcttaccgggttggac
tcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttgga-
gcgaacgacctaca
ccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtat-
ccggtaagcggcag
ggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttc-
gccacctctgacttgag
cgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggtt-
cctggccttttgctggcctt
ttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtgcggccgccccttgtaATTCtatg-
agcttatgtacatcaaAt
acagcggccgcgattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatct-
aaagtatatatgagtaaacttg
gtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttg-
cctgactccccgtcgtgtagat
aactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgggacccacgctcaccggctc-
cagatttatcagcaata
aaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattg-
ttgccgggaagctagag
taagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcg-
tttggtatggcttcattcagct
ccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcct-
ccgatcgttgtcagaagta
agttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaaga-
tgcttttctgtgactggtgagta ctcaaccaagtcattctgagaatagtgtatgcggcg (SEQ
ID NO: 104)
Example 4
Results
[0694] Transcriptomic mapping suggested that crRNA was expressed
heterologously in E. coli cells and processed to include a 25
nucleotide-long repeat and a 14-20 nucleotide spacer. The data also
suggested that Cas12J likely processes its own crRNA (see FIG.
14A-14C),
[0695] FIG. 14A-14C illustrates results from mapping RNA sequences
to the Cas12J CRISPR locus from pBAS::Cas12J-1947455 (FIG. 14A),
pBAS::Cas12J-2071242 (FIG. 14B), and pBAS::Cas12J-3339380 (FIG.
14C). Inset shows a detailed view of transcriptome mapping to the
first repeat-spacer-repeat iteration in each locus. Black diamonds
denote repeats; colored squares denote spacers; faded repeats and
spacers denote the degenerate end of the array.
Methods
RNA-seq
[0696] pBAS::Cas12J-1947455, pBAS::Cas12J-2071242, and
pBAS::Cas12J-3339380 constructs were transformed in chemically
competent E. coli DH5a (QB3-Macrolab, UC Berkeley) and incubated
over night at 37.degree. C. on LB-Cm agar plates (34 .mu.g/mL
chloramphenicol). Single colonies were picked to inoculate 5 mL
(LB, 34 .mu.g/mL chloramphenicol) starter cultures which were
incubated at 37.degree. C. shaking vigorously overnight. The next
morning, main cultures were inoculated 1:100 (LB, 34 .mu.g/mL
chloramphenicol) and locus expression was induced with 200 nM aTc
for 24 h at 16.degree. C. Cells were harvested by centrifugation,
resuspended in lysis buffer (20 mM Hepes-Na pH 7.5, 200 mM NaCl)
and lysed using glass beads (0.1 mm glass beads, 4.times.30 s
vortex at 4.degree. C., interspaced by 30 s cool-down on ice). 200
.mu.L cell lysis supernatant were transferred into Trizol for RNA
extraction according to the manufacturers protocol (Ambion). 10
.mu.g RNA were treated with 20 units of T4-PNK (NEB) for 6 h at
37.degree. C. for dephosphorylation. Subsequently, 1 mM ATP was
added and the sample was incubated for 1 h at 37.degree. C. for
5'-phosphorylation before heat inactivation at 65.degree. C. and
subsequent Trizol purification.
[0697] Next, cDNA libraries were prepared using the RealSeq-AC
miRNA library kit illumina sequencing (somagenics). cDNA libraries
were subjected to Illumina MiSeq sequencing, generating 50
nucleotide-long single reads. Raw sequencing data was processed to
remove adapters and sequencing artifacts, and high-quality reads
were maintained. The resulting reads were mapped to their
respective plasmids to determine the CRISPR locus expression and
crRNA processing.
Example 5
Results
[0698] The data provided in FIG. 15 show that Cas12J can induce
targeted GFP disruption, indicating successful Non-Homologous End
Joining (NHEJ) and targeted genomic editing in human cells. In one
case, an individual Cas12J/guide RNA was able to edit as high as
33% of cells (Cas12J-2 guide 2), comparable to levels reported for
CRISPR-Cas9. CRISPR-Cas12a, and CRISPR-CasX (Gong et al. (2013)
Science 339:819; Jinek et al. (2013) eLife 2:e00471; Mali et al.
(2013) Science 339; 823; and Liu et al, (2019) Nature
566:7743).
Methods
Cloning of Cas12J Effector Plasmids for Expression in Human
Cell
[0699] The gene sequence of cas12J-2 and cas12J-3 were ordered as
G-blocks from Integrated DNA Technologies (IDT) encoding codon
optimized genes for expression in human cells. G-blocks were cloned
via Golden Gate assembly into the vector backbone of pBL062.5,
downstream fused to two SV40 NLSs via a GSG linker encoding
sequence (FIG. 16A-16B, providing construct maps; and Table 1
(provided in FIG. 17A-17G), providing nucleotide sequences of the
constructs). The guide encoding sequence of pBL062.5 was exchanged
to encode for a single CRISPR-repeat of the respective homologue,
followed by a 20 bp stuffer spacer sequence amenable to Golden Gate
exchange using the restriction enzyme SapI (FIG. 16A-16B; and Table
1 (provided in FIG. 17A-17G)). To generate EGFP targeting
constructs, the stuffer was exchanged via Golden Gate assembly to
encode the guide for the selected target site (Table 2).
TABLE-US-00016 TABLE 2 Guide sequences Guide # Spacer Sequence
5'.fwdarw.3' NT CGTGATGGTCTCGATTGAGT (SEQ ID NO: 105) 1
ACCGGGGTGGTGCCCATCCT (SEQ ID NO: 106) 2 ATCTGCACCACCGGCAAGCT (SEQ
ID NO: 107) 3 GAGGGCGACACCCTGGTGAA (SEQ ID NO: 108)
Human-Cell Targeted GFP Disruption
[0700] The GFP HEK293 reporter cells were previously generated via
lentiviral integration as previously described. Antony et al.
(2018) Mol. Cell. Pediatrics 5:9. Cells were routinely tested for
mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza),
according to the manufacturer's protocol, GFP HEK293 reporter cells
were seeded into 96-well plates and transfected the next day with
lipofectamine 3000 (Life Technologies) and 200 ng of plasmid DNA
encoding the Cas12J gRNA and Cas12J-P2A-puromycin fusion. 24 hours
post-transfection, successfully transfected cells were selected for
by adding 1.5 .mu.g/mL puromycin to the cell culture media for 72
hours, Cells were passaged to maintain sub-confluent conditions and
then analyzed on an Attune N.times.T Flow Cytometer with an
autosampler. Cells were analyzed on the flow cytometer after 7 days
to allow for clearance of GFP from cells.
Example 6
Results
[0701] To test whether Cas12J features an unspecific trans-cleavage
activity, once activated by cis-targeted nucleic acids, an in vitro
cleavage assay was set up. In the assay, the Cas12J RNPs and trans
cleavage ssDNA or ssRNA substrates were incubated in the presence
of no cis-activator, ssDNA cis-activator, dsDNA cis-activator, or
ssRNA cis-activator.
[0702] As shown in FIG. 18, the three tested Cas12J homologs
efficiently cleave ssDNA, but not ssRNA, when an activating DNA,
but not RNA, is present in the reaction. This assay demonstrates
that Cas12J can be activated by spacer complementary ssDNA, or
dsDNA, to target ssDNA in trans. Furthermore, this DNA-activated
ssDNA trans cleavage activity can be used for nucleic acid
detection using a Fluorophore-quencher labeled reporter assay
(East-Seletsky et al., Nature 538, 270-273 (2016)).
Methods
[0703] ssDNA and ssRNA substrates for trans cleavage were designed
to be non-complementary to the spacer of the Cas12J guide RNA.
Substrates were 5'-end-labelled using T4-PNK (NEB) in the presence
of .sup.32P-.gamma.-ATP. Active Cas12J RNP complexes were assembled
by diluting Cas12J protein and guide crRNA to 4 .mu.M in complex
assembly buffer (20 mM HEPES-Na pH 7.5 RT, 300 mM KCl, 10 mM
MgCl.sub.2, 20% glycerol, 1 mM TCEP) and incubation for 30 min at
RT. Spacer complementary activator substrates were diluted in
oligonucleotide hybridization buffer (10 mM Tris pH 7.8 RT, 150 mM
KCl) to a concentration of 4 .mu.M, heated to 95.degree. C. for 5
min, and subsequently cooled down at room temperature (RT) to allow
duplex formation for double stranded activator substrates. Cleavage
reactions were set up by combining 200 nM RNP with 400 nM activator
substrate and incubation for 10 min at RT before addition of 2 nM
ssDNA, or ssRNA, trans cleavage substrates. Reactions were
conducted in reaction buffer (10 mM HEPES-Na pH 7.5 RT, 150 mM KCl,
5 mM MgCl.sub.2, 10% glycerol, 0.5 mM TCEP) and incubated for 60
min at 37.degree. C. Reactions were stopped by addition of two
volumes formamide loading buffer (96% formamide, 100 .mu.g/mL
bromophenol blue, 50 .mu.g/mL xylene cyanol, 10 mM EDTA, 50
.mu.g/mL heparin), heated to 95.degree. C. for 5 min, and cooled
down on ice before separation on a 12.5% denaturing
urea-polyacrylamide gel electrophoresis (PAGE). Gels were dried for
4 h at 80.degree. C. before phosphor-imaging visualization using an
Amersham Typhoon scanner (GE Healthcare).
Example 7
Materials and Methods
Metagenomic Assemblies, Genome Curation, and CRISPR-Cas.PHI.
(CRISPR-Cas12J) Detection
[0704] Metagenomic sequencing data was assembled using previously
described methods (Peng et al. Bioinformatics. 28, 1420-1428
(2012); and Nurk et al. Genome Res. 27, 824-834 (2017). Coding
sequences (CDS) were predicted from sequence assemblies using
prodigal with genetic code 11 (-m -g 11 -p single) and (-m -g 11 -p
meta) and preliminary annotations were performed as previously
described by searching against UniProt, UniRef100, and KEGG
(Wrighton et al, ISME J. 8, 1452-1463 (2014)). Phage genome
curation was performed as described above. Briefly, Bowtie2
v2.3.4.1 (Langmead and Salzberg Nat. Methods. 9, 357-359 (2012))
was used to map reads to the de novo assembled sequences, and
unplaced mate pairs of mapped reads were retained with shrinksam
(github.com/bcthomas/shrinksam). N-filled gaps and local
misassemblies were identified and corrected, and unplaced or
incorrectly placed paired reads allowed extension of contig ends.
Local assembly changes and extensions were verified with further
read mapping. A database of Cas.PHI. sequences was generated using
MAFFT v7.407 (Katoh and Standley Mol. Biol. Evol. 30, 772-780
(2013)) and hmmbuild. CDS from new assemblies were searched against
the HMM database using hmmsearch with e-value <1.times.10.sup.-5
and added to the database upon verification.
Phylogenetic Analysis of Type V Systems
[0705] Cas protein sequences were collected as described above and
representatives from the TnpB superfamily were collected from
Makarova et al. (Nat. Rev. Microbiol., 1-17 (2019)) and top BLAST
hits from RefSeq. The resulting set was clustered at 90% amino acid
identity using CD-HIT to reduce redundancy (Huang et al.
Bioinformatics. 26, 680-682 (2010)). A new alignment of Cas.PHI.,
with the resulting sequence set was generated using MAFFT LINSI
with 1000 iterations and filtered to remove columns comprised of
gaps in 95% of sequences. Poorly aligned sequences were removed and
the resulting set was realigned. The phylogenetic tree was inferred
using IQTREE v1.6.6 using automatic model selection (Nguyen et al.
Mol. Biol. Evol. 32, 268-274 (2015)) and 1000 bootstraps.
crRNA Sequence Analysis
[0706] CRISPR-RNA (crRNA) repeats from Phage-encoded CRISPR loci
were identified using MinCED (github.com/ctSkennerton/minced) and
CRISPRDetect (Biswas et al. BMC Genomics. 17, 356 (2016)). The
repeats were compared by generating pairwise similarity scores
using the Needleman-Wunsch algorithm followed by EMBOSS Needle
(McWilliam et al. Nucleic Acids Res. 41, W597-600 (2013)). A
heatmap was built using the similarity score matrix and
hierarchical clustering produced dendrograms that were overlaid
onto the heatmap to delineate different clusters of repeats.
Generation of Plasmids
[0707] Cas.PHI. loci, including an additional E. coli RBS upstream
of cas.PHI., were ordered as G-blocks from Integrated DNA
Technologies (IDT) and cloned using Golden Gate assembly (GG) under
the control of a tetracycline-inducible promoter for RNA seq and
PAM depletion plasmid interference experiments. Perfect
repeat-spacer units of the CRISPR-arrays identified by metagenomics
were reduced to a single repeat-spacer-repeat unit, amenable to
stuffer-spacer exchange by GG-assembly (AarI-restriction sites).
Subsequently, Cas.PHI. gene sequences were subcloned by GG-assembly
into pRSFDuet-1 (Novagen) within MCSI without tags for efficiency
of transformation plasmid interference assays, or fused to a
C-terminal hexa-histidine tag for protein purification. For plasmid
interference assays, mini-CRISPR arrays (repeat-spacer-repeat, or
repeat-spacer-HDV ribozyme) amenable to stuffer-spacer exchange by
GG-assembly (AarI-restriction sites) were cloned into MCS II of
pRSFDuet. For genome editing experiments in human cells, cas.PHI.
genes were ordered as G-blocks from IDT encoding codon optimized
genes for expression in human cells. G-blocks were cloned via
GG-assembly into the vector backbone of pBL062.5, downstream fused
to two SV40 NLSs via a GSG linker encoding sequence. The guide
encoding sequence of pBL062.5 was exchanged to encode for a single
CRISPR-repeat of the respective homologue, followed by a 20 bp
stuffer spacer sequence amenable to GG-assembly exchange using the
restriction enzyme SapI. A list of plasmids and a brief description
is given in FIG. 34 (providing Table 3). Plasmid sequences and maps
will be made available on addgene. To reprogram the Cas.PHI.
vectors to target different loci, stuffer-spacer were exchanged via
GG-assembly to encode the guide for the selected target site (guide
spacer sequences are listed in FIG. 35 (providing Table 4)).
Mutations in the cas.PHI. genes were introduced by GG-assembly to
create dcas.PHI. genes.
PAM Depletion DNA Interference Assay
[0708] PAM depletion assays were performed with both, Cas.PHI.
plasmids that either carried the whole Cas locus as derived from
metagenomics (pPP049, pPP056 and pPP062), or with plasmids that
contained only the cas.PHI. gene and a mini CRISPR (pPP097, pPP102
and pPP107). Assays were performed as three individual biological
replicates. Plasmids containing cas.PHI. and mini CRISPRs were
transformed into E. coli BL21(DE3) (NEB) and constructs containing
Cas.PHI. genomic loci were transformed into E. coli DH5a
(QB3-Macrolab, UC Berkeley). Subsequently, electrocompetent cells
were prepared by ice cold H.sub.2O and 10% glycerol washing. A
plasmid library was constructed with 8 randomized nucleotides
upstream (5') end of the target sequence. Competent cells were
transformed in triplicate by electroporation with 200 ng library
plasmids (0.1 mm electroporation cuvettes (Bio-Rad) on a
Micropulser electroporator (Bio-Rad)). After a two-hour recovery
period, cells were plated on selective media and colony forming
units were determined to ensure appropriate coverage of all
possible combinations of the randomized 5' PAM region. Strains were
grown at 25.degree. C. for 48 hours on media containing appropriate
antibiotics (either 100 .mu.g/mL carbenicillin and 34 .mu.g/mL
chloramphenicol, or 100 .mu.g/mL carbenicillin and 50 .mu.g/mL
kanamycin) and 0.05 mM isopropyl-.beta.-D-thiogalactopyranoside
(IPTG), or 200 nM anhydrotetracycline (aTc), depending on the
vector to ensure propagation of plasmids and Cas.PHI. effector
production. Subsequently, propagated plasmids were isolated using a
QIAprep Spin Miniprep Kit (Qiagen).
PAM Depletion Sequencing Analysis
[0709] Amplicon sequencing of the targeted plasmid was used to
identify PAM motifs that are preferentially depleted. Sequencing
reads were mapped to the respective plasmids and PAM randomized
regions were extracted. The abundance of each possible 8 nucleotide
combination was counted from the aligned reads and normalized to
the total reads for each sample. Enriched PAMs were computed by
calculating the log ratio compared to the abundance in the control
plasmids, and were used to produce sequence logos.
RNA Preparation for RNAseq
[0710] Plasmids containing Cas.PHI. loci were transformed into
chemically competent E. coli DH5.alpha. (QB3-Macrolab, UC
Berkeley). Preparations were performed as three individual
biological replicates. Single colonies were picked to inoculate 5
mL starter cultures (LB, 34 .mu.g/mL chloramphenicol) which were
incubated at 37.degree. C. shaking vigorously overnight. The next
morning, main cultures were inoculated 1:100 (LB, 34 .mu.g/mL
chloramphenicol) and locus expression was induced with 200 nM aTc
for 24 h at 16.degree. C. Cells were harvested by centrifugation,
resuspended in lysis buffer (20 mM Hepes-Na pH 7.5 RT, 200 mM NaCl)
and lysed using glass beads (0.1 mm glass beads, 4.times.30 s
vortex at 4.degree. C., interspaced by 30 s cool-down on ice). 200
.mu.L cell lysis supernatant were transferred into Trizol for RNA
extraction according to the manufacturer's protocol (Ambion). 10
.mu.g RNA were treated with 20 units of T4-PNK (NEB) for 6 h at
37.degree. C. for 2'-3'-dephosphorylation. Subsequently, 1 mM ATP
was added and the sample was incubated for 1 h at 37.degree. C. for
5'-phosphorylation before heat inactivation at 65.degree. C. for 20
min and subsequent Trizol purification.
RNA Analysis by RNAseq
[0711] cDNA libraries were prepared using the RealSeq-AC miRNA
library kit illumina sequencing (somagenics). cDNA libraries were
subjected to Illumina MiSeq sequencing, and raw sequencing data was
processed to remove adapters and sequencing artifacts, and
high-quality reads were maintained. The resulting reads were mapped
to their respective plasmids to determine the CRISPR locus
expression and crRNA processing, and coverage was calculated at
each region.
Efficiency of Transformation Plasmid Interference Assay
[0712] Cas.PHI. vectors were transformed into chemically competent
E. coli BL21(DE3) (NEB). Individual colonies for biological
replicates were picked to inoculate three 5 mL (LB, Kanamycin 50
.mu.g/mL) starter cultures to prepare electrocompetent cells the
following day. 50 mL (LB, Kanamycin 50 .mu.g/mL) main cultures were
inoculated 1:100 and grown vigorously shaking at 37.degree. C. to
an OD.sub.600 of 0.3. Subsequently, the cultures were cooled to
room temperature and case expression was induced with 0.2 mM IPTG.
Cultures were grown to an OD.sub.600 of 0.6-0.7 at 25.degree. C.,
before preparation of electrocompetent cells by repeated ice-cold
H.sub.2O and 10% glycerol washes. Cells were resuspended in 250
.mu.L 10% glycerol. 90 .mu.L aliquots were flash frozen in liquid
nitrogen and stored at -80.degree. C. The next day, 80 .mu.L
competent cells were combined with 3.2 .mu.L plasmid (20 ng/.mu.L
pUC19 target plasmid, or 20 ng/.mu.L pYTK001 control plasmid),
incubated for 30 min on ice and split into three individual 25
.mu.L transformation reactions. After electroporation in 0.1 mm
electroporation cuvettes (Bio-Rad) on a Micropulser electroporator
(Bio-Rad), cells were recovered in 1 mL recovery medium (Lucigen)
supplemented with 0.2 mM IPTG, shaking at 37.degree. C. for one
hour. Subsequently, 10-fold dilution series were prepared and 5
.mu.L of the respective dilution steps were spot-plated on LB-Agar
containing the appropriate antibiotics. Plates were incubated
overnight at 37.degree. C. and colonies were counted the following
day to determine the transformation efficiency. To assess the
transformation efficiency, the mean and standard deviations were
calculated from the cell forming units per ng transformed plasmids
for the electroporation triplicates.
Protein Production and Purification
[0713] Cas.PHI. overexpression vectors were transformed into
chemically competent E. coli BL21(DE3)-Star (QB3-Macrolab, UC
Berkeley) and incubated overnight at 37.degree. C. on LB-Kan agar
plates (50 .mu.g/mL Kanamycin). Single colonies were picked to
inoculate 80 mL (LB, Kanamycin 50 .mu.g/mL) starter cultures which
were incubated at 37.degree. C. shaking vigorously overnight. The
next day, 1.5 L TB-Kan medium (50 .mu.g/mL Kanamycin) were
inoculated with 40 mL starter culture and grown at 37.degree. C. to
an OD.sub.600 of 0.6, cooled down on ice for 15 min and gene
expression was subsequently induced with 0.5 mM IPTG followed by
incubation overnight at 16.degree. C. Cells were harvested by
centrifugation and resuspended in wash buffer (50 mM HEPES-Na pH
7.5 RT, 1 M NaCl, 20 mM imidazole, 5% glycerol and 0.5 mM TCEP),
subsequently lysed by sonication, followed by lysate clarification
by centrifugation. The soluble fraction was loaded on a 5 mL Ni-NTA
Superflow Cartridge (Qiagen) pre-equilibrated in wash buffer. Bound
proteins were washed with 20 column volumes (CV) wash buffer and
subsequently eluted in 5 CV elution buffer (50 mM HEPES-Na pH 7.5
RT, 500 mM NaCl, 500 mM imidazole, 5% glycerol and 0.5 mM TCEP).
The eluted proteins were concentrated to 1 mL before injection into
a HiLoad 16/600 Superdex 200 pg column (GE Healthcare)
pre-equilibrated in size-exclusion chromatography buffer (20 mM
HEPES-Na pH 7.5 RT, 500 mM NaCl, 5% glycerol and 0.5 mM TCEP). Peak
fractions were concentrated to 1 mL and concentrations were
determined using a NanoDrop 8000 Spectrophotometer (Thermo
Scientific). Proteins were purified at a constant temperature of
4.degree. C. and concentrated proteins were kept on ice to prevent
aggregation, snap frozen in liquid nitrogen and stored at
-80.degree. C. AsCas12a was purified as previously described (Knott
et al. (2019) Nat. Struct. Mol. Biol. 26:315).
In Vitro Cleavage Assays--Spacer Tiling
[0714] Plasmid targets were cloned by GG-assembly of spacer 2,
found in the CRISPR-array of Cas.PHI.-1, downstream to a cognate
5'-TTA PAM, or non-cognate 5'-CCA PAM into pYTK095 (Target
sequences are given in FIG. 36 (providing Table 5)). Supercoiled
plasmids were prepared by propagation of the plasmid overnight at
37.degree. C. in E. coli Mach1 (QB3-Macrolab, UC Berkeley) in LB
and Carbenicillin (100 .mu.g/mL) and subsequent preparation using a
Qiagen Miniprep kit (Qiagen). Linear DNA targets were prepared by
PCR from the plasmid target. crRNA guides were ordered as synthetic
RNA oligos from IDT (FIG. 37 (providing Table 6)), dissolved in
DEPC H.sub.20 and heated for 3 min at 95.degree. C. before cool
down at RT. Active RNP complexes were assembled at a concentration
of 1.25 .mu.M by mixing protein and crRNA (IDT) in a 1:1 molar
ratio in cleavage buffer (10 mM Hepes-K pH 7.5 RT, 150 mM KCl, 5 mM
MgCl.sub.2, 0.5 mM TCEP) and incubation at RT for 30 min. Cleavage
reactions were initiated by addition of DNA (10 nM) to preformed
RNP (1 .mu.M) in reaction buffer (10 mM Hepes-K pH 7.5 RT, 150 mM
KCl, 5 mM MgCl.sub.2, 0.5 mM TCEP). The reactions were incubated at
37.degree. C., quenched with 50 mM EDTA and stored in liquid
nitrogen. Samples were thawed and treated with 0.8 units proteinase
K (NEB) for 20 min at 37.degree. C. Loading dye was added (Gel
Loading Dye Purple 6.times., NEB) and samples were analyzed by
electrophoresis on a 1% agarose gel and stained with SYBR Safe
(Thermo Fisher Scientific). For comparison to cleavage products,
supercoiled plasmids were digested with PciI (NEB) for
linearization and Nt.BstNBI (NEB) for plasmid nicking and open
circle formation. Comparable cleavage assays under varied
conditions (n.gtoreq.3) showed consistent results.
In Vitro Cleavage Assays--Radiolabeled Nucleic Acids
[0715] Active Cas.PHI. RNP complexes were assembled in a 1:1.2
molar ratio by diluting Cas.PHI. protein to 4 .mu.M and crRNA (IDT)
to 5 .mu.M in RNP assembly buffer (20 mM HEPES-Na pH 7.5 RT, 300 mM
KCl, 10 mM MgCl.sub.2, 20% glycerol, 1 mM TCEP) and incubation for
30 min at RT. Substrates were 5'-end-labelled using T4-PNK (NEB) in
the presence of .sup.32P-.gamma.-ATP (Substrate sequences are given
in FIG. 36 (providing Table 5)). Oligo-duplex targets were
generated by combining .sup.32P-labelled and unlabeled
complementary oligonucleotides in a 1:1.5 molar ratio. Oligos were
hybridized to a DNA-duplex concentration of 50 nM in hybridization
buffer (10 mM Tris-Cl pH 7.5 RT, 150 mM KCl), by heating for 5 min
to 95.degree. C. and a slow cool down to RT in a heating block.
Cleavage reactions were initiated by combining 200 nM RNP with 2 nM
substrate in reaction buffer (10 mM HEPES-Na pH 7.5 RT, 150 mM KCl,
5 mM MgCl.sub.2, 10% glycerol, 0.5 mM TCEP) and subsequently
incubated at 37.degree. C. For trans-cleavage assays, guide
complementary activator substrates were diluted in oligonucleotide
hybridization buffer (10 mM Tris pH 7.8 RT, 150 mM KCl) to a
concentration of 4 .mu.M, heated to 95.degree. C. for 5 min, and
subsequently cooled down at RT to allow duplex formation for double
stranded activator substrates. Cleavage reactions were set up by
combining 200 nM RNP with 100 nM activator substrate and incubation
for 10 min at RT before addition of 2 nM ssDNA, or ssRNA, trans
cleavage substrates. Reactions were stopped by addition of two
volumes formamide loading buffer (96% formamide, 100 .mu.g/mL
bromophenol blue, 50 .mu.g/mL xylene cyanol, 10 mM EDTA, 50
.mu.g/mL heparin), heated to 95.degree. C. for 5 min, and cooled
down on ice before separation on a 12.5% denaturing urea-PAGE. Gels
were dried for 4 h at 80.degree. C. before phosphor-imaging
visualization using an Amersham Typhoon scanner (GE Healthcare).
Technical replicates (n.gtoreq.2) and comparable cleavage assays
under varied conditions (n.gtoreq.3) of biological replicates
(n.gtoreq.2) showed consistent results. Bands were quantified using
ImageQuant TL (GE) and cleaved substrate was calculated from the
intensity relative to the intensity observed at t=0 min. Curves
were fit to a One-Phase-Decay model in Prism 8 (graphpad) to derive
the rate of cleavage.
In Vitro Pre-crRNA Processing Assay
[0716] Pre-crRNA substrates were 5'-end-labelled using T4-PNK (NEB)
in the presence of .sup.32P-.gamma.-ATP (Substrate sequences are
given in FIG. 36 (providing Table 5)). Processing reactions were
initiated by combining 50 nM Cas.PHI. with 1 nM substrate in
pre-crRNA processing buffer (10 mM Tris pH 8 RT, 200 mM KCl, 5 mM
MgCl.sub.2 or 25 mM EDTA, 10% glycerol, 1 mM DTT) and subsequently
incubated at 37.degree. C. Substrate hydrolysis ladders were
prepared using the alkaline hydrolysis buffer according to the
manufacturer's protocol (Ambion). 10 .mu.L of the processing
reaction products were treated with 10 units T4-PNK (NEB) for 1 h
at 37.degree. C. in the absence of ATP for termini chemistry
analysis. Reactions were stopped by addition of two volumes
formamide loading buffer (96% formamide, 100 .mu.g/mL bromophenol
blue, 50 .mu.g/mL xylene cyanol, 10 mM EDTA, 50 .mu.g/mL heparin),
heated to 95.degree. C. for 3 min, and cooled down on ice before
separation on a 12.5%, or 20%, denaturing urea-PAGE. Gels were
dried for 4 h at 80.degree. C. before phosphor-imaging
visualization using an Amersham Typhoon scanner (GE Healthcare).
Technical replicates (n.gtoreq.3) and comparable cleavage assays
under varied conditions (n.gtoreq.3) of biological replicates
(n.gtoreq.2) showed consistent results. Bands were quantified using
ImageQuant TL (GE) and processed RNA was calculated from the
intensity at t=60 min relative to the intensity observed at t=0
min.
Analytical Size Exclusion Chromatography
[0717] 500 .mu.L samples (5-10 .mu.M protein, RNA, or reconstituted
RNPs) were injected onto a S200 XK10/300 size exclusion
chromatography (SEC) column (GE Healthcare) pre-equilibrated in SEC
buffer (20 mM HEPES-Cl pH 7.5 RT, 250 mM KCl, 5 mM MgCl.sub.2, 5%
glycerol and 0.5 mM TCEP). Prior to SEC, Cas.PHI. RNP complexes
were assembled by incubating Cas.PHI. protein and pre-crRNA for 1 h
in 2.times. pre-crRNA processing buffer (20 mM Tris pH 7.8 RT, 400
mM KCl, 10 mM MgCl.sub.2, 20% glycerol, 2 mM DTT).
Genome Editing in Human Cells
[0718] The GFP HEK293 reporter cells were generated via lentiviral
integration as previously described. Richardson et al. (2016) Nat.
Biotechnol. 34:339. Cells were routinely tested for absence of
mycoplasma using the MycoAlert Mycoplasma. Detection Kit (Lonza),
according to the manufacturer's protocol. GFP HEK293 reporter cells
were seeded into 96-well plates and transfected at 60-70%
confluency the next day according to the manufacturer's protocol
with lipofectamine 3000 (Life Technologies) and 200 ng of plasmid
DNA encoding the Cas.PHI. gRNA and Cas.PHI.-P2A-PAC fusion. As a
comparison control, 2.00 ng of plasmid DNA encoding the SpyCas9
sgRNA and SpyCas9-P2A-PAC fusion was transfected identically, with
target sequences adjusted for PAM differences. 24 hours
post-transfection., successfully transfected cells were selected
for by addling 1.5 ug/mL puromycin to the cell culture media for 72
hours. Cells were passaged regularly to maintain sub-confluent
conditions and then analyzed on an Attune NxT Flow Cytometer with
an autosampler. Cells were analyzed on the flow cytometer after 10
days to allow for clearance of GFP from cells.
Results
[0719] Cas12J, or simply Cas.PHI. as homage to its phage-restricted
origin, is a previously unknown family of Cas proteins encoded in
the Biggiephage clade. Cas.PHI. contains a C-terminal RuvC domain
with remote homology to that of the TnpB nuclease superfamily from
which type V CRISPR-Cas proteins are thought to have evolved (FIG.
20). However, Cas.PHI. shares <7% amino acid identity with other
type V CRISPR-Cas proteins and is most closely related to a TnpB
group distinct from miniature type V (Cas14) proteins (FIG.
19A).
[0720] Cas.PHI.'s unusually small size of .about.70-80 kDa, about
half the size of the RNA-guided DNA cutting enzymes Cas9 and Cas12a
(FIG. 19B), and its lack of co-occurring genes raised the question
of whether Cas.PHI. functions as a bona fide CRISPR-Cas system.
Three different Cas.PHI. orthologs from metagenomic assemblies were
selected for study based on divergence of their protein and CRISPR
repeat sequences (FIG. 21), referred to in FIG. 21 as Cas.PHI.-1,
Cas.PHI.-2 and Cas.PHI.-3. To investigate the ability of Cas.PHI.
to recognize and target DNA in bacterial cells, it was tested
whether these systems could protect Escherichia coli from plasmid
transformation. CRISPR-Cas systems are known to target DNA
sequences following or preceding a 2-5 nucleotide Protospacer
Adjacent Motif (PAM) for self-versus-non-self discrimination
(Gleditzsch et al. (2019) RNA Biology 16:504). To determine whether
Cas.PHI. uses a PAM, a library of plasmids containing randomized
regions adjacent to crRNA-complementary target sites was
transformed into E. coli, thereby preferentially depleting plasmids
including functional PAMs. This revealed the crRNA-guided
double-strand DNA (dsDNA) targeting capability of Cas.PHI. and
distinct T-rich PAM sequences, including a minimal 5'-TBN-3' PAM
observed for Cas.PHI.-2 (FIG. 19C).
[0721] The E. coli expression system and plasmid interference assay
was used to determine the components required for CRISPR-Cas.PHI.
system function. RNA-sequencing analysis revealed transcription of
the case gene and CRISPR array but no evidence of other non-coding
RNA such as a trans-activating CRISPR RNA (tracrRNA) encoded in or
near the locus (FIG. 19D). In addition, it was found that Cas.PHI.
activity could be readily directed against other plasmid sequences
by altering the guide RNA, demonstrating the programmability of
this system (FIG. 22A-22C). These findings suggest that in its
native environment, Cas.PHI. is a functional phage protein and bona
fide CRISPR-Cas effector capable of cleaving DNA bearing
complementarity to different crRNAs, likely other MGEs, to abrogate
superinfection (FIG. 19E). Furthermore, these results demonstrate
that this single-RNA system is much more compact than other active
CRISPR-Cas systems (FIG. 19F).
[0722] CRISPR-Cas effector complexes identify and cleave foreign
nucleic acids during the final stage of CRISPR-Cas mediated
immunity against MGEs (Hille et al. (2018) Cell 172:1239). To
determine how Cas.PHI., achieves RNA-guided DNA targeting for
Biggiephages, the recognition and cleavage requirements of Cas.PHI.
in vitro were investigated. RNA-seq revealed that the spacer
sequence within the crRNA, which is complementary to DNA targets,
is between 14-20 nucleotides (nt) long (FIG. 19D). Incubation of
purified Cas.PHI. (FIG. 24A-24D) with crRNAs of different spacer
sizes along with supercoiled plasmid or linear dsDNA revealed that
target DNA cleavage requires the presence of a cognate PAM and a
spacer of >14 nt (FIG. 23A; FIG. 25A). Analysis of the cleavage
products showed that Cas.PHI. generates staggered 5'-overhangs of
8-12 nt (FIGS. 23B and 23C; FIGS. 25B and 25C), similar to the
staggered DNA cuts observed for other type V CRISPR-Cas enzymes
including Cas12a and CasX (Zetsche et al. (2015) Cell 163:759; Liu
et al. (2019) Nature 566:218). It was observed that Cas.PHI.-2 and
Cas.PHI.-3 were more active in vitro than Cas.PHI.-1, and the
non-target strand (NTS) was cleaved faster than the target-strand
(TS) (FIG. 23D; FIG. 26A; FIGS. 27A and 27B). Furthermore, Cas.PHI.
was found to cleave ssDNA but not ssRNA targets (FIG. 26B),
suggesting that Cas.PHI. may also target ssDNA MGEs or ssDNA
intermediates.
[0723] To assess the role of the RuvC domain in Cas.PHI.-catalyzed
DNA cleavage, the active site was mutated (D371A, D394A, or D413A)
to produce a Cas.PHI., variant (dCas.PHI.) that was found not to
cleave dsDNA, ssDNA or ssRNA in vitro (FIGS. 26A and 26B). When
expressed in E. coli along with the CRISPR array, dCas.PHI. could
not prevent transformation of a crRNA-complementary plasmid,
consistent with a requirement for RuvC-catalyzed DNA cutting (FIG.
22A-22B). This observation, together with the delayed cleavage of
the target strand after non-target strand cleavage (FIG. 23D; FIGS.
27A and 27B), suggests that Cas.PHI. cleaves each strand
sequentially within the RuvC active site. Sequential dsDNA strand
cleavage is consistent with the dsDNA cutting mechanism of the type
V CRISPR-Cas proteins (10) that share closest evolutionary origin
with Cas.PHI..
[0724] Furthermore, like other type V CRISPR-Cas effectors,
Cas.PHI. was found to degrade ssDNA in trans when activated by
target dsDNA or ssDNA binding in cis. Trans single-stranded DNAse,
but not RNAse, activity upon DNA target recognition in cis was
observed (FIG. 28A-28B). This trans-cleavage activity, coupled with
a minimal PAM requirement, may be useful for broader nucleic acid
detection.
[0725] To provide genome defense, CRISPR-Cas.PHI. systems must
produce mature crRNA transcripts to guide foreign DNA cleavage.
Other type V CRISPR-Cas proteins process their own pre-crRNAs using
an internal active site distinct from the RuvC domain (Fonfara et
al. Nature. 532, 517-521 (2016)) or by recruiting Ribonuclease III
to cleave a duplex RNA substrate formed by pre-crRNA base pairing
with a tracrRNA (Burstein et al. (2017) Nature 542:237; Harrington
et al. (2018) Science 362:839; Yan et al. (2019) Science 363:88;
Shmakov et al. (2015) Mol. Cell. 60:385). The absence of a
detectable tracrRNA encoded in CRISPR-Case genomic loci hinted that
Cas.PHI. may catalyze crRNA maturation on its own. To test this
possibility, purified Cas.PHI. was incubated with substrates
designed to mimic the pre-crRNA structure (FIG. 29A). Reaction
products corresponding to a 26-29 nucleotide-long repeat and 20
nucleotide guide sequence of the crRNA were observed only in the
presence of wildtype Case, corroborated by RNA-seq analysis of
native loci (FIG. 19D; FIG. 29A; FIG. 29C; FIG. 30A-30C). In
control experiments, it was found that Case-catalyzed pre-crRNA
processing is magnesium-dependent (FIG. 29B; FIG. 30A-30C), which
is different from all other known CRISPR-Cas RNA processing
reactions and suggested a distinct chemical mechanism of cleavage.
Notably, the RuvC domain itself employs a magnesium-dependent
mechanism to cleave DNA substrates (Nowotny et al. (2009) EMBO Rep.
10:144), and some RuvC domains have been reported to have
endoribonucleolytic activity (Yan et al. (2019) Science 363:88).
Based on these observations, a Cas.PHI. containing a
RuvC-inactivating mutation was tested; it was found to be incapable
of processing pre-crRNAs (FIG. 29B; FIGS. 30A and 30B). Both
wild-type and catalytically inactivated Cas.PHI. proteins are
capable of crRNA binding, and their reconstituted complexes with
pre-crRNA have similar elution profiles from a size exclusion
column, suggesting no pre-crRNA binding or protein stability defect
resulting from the RuvC point mutation (FIG. 31A-31B).
[0726] It was hypothesized that if the Cas.PHI. RuvC domain is
responsible for pre-crRNA cleavage, the products should contain
5'-phosphate and 2'- and 3'-hydroxyl moieties as observed in RNAs
generated by the RuvC-related RNase HI enzymes (Nowotny et al.
(2009) supra). In contrast, other type V CRISPR-Cas enzymes process
pre-crRNA by a metal-independent acid-base catalysis mechanism in
an active site distinct from the RuvC domain, generating
2'-3'-cyclic phosphate crRNA termini, as observed for Cas12a
(Swarts et al. (2017) Mol. Cell. 66:221). PNK phosphatase treatment
of Case-generated crRNA followed by denaturing acrylamide gel
analysis showed no change in the crRNA migration behavior, distinct
from the change in mobility detected in a similar experiment
conducted with crRNA generated by Cas12a (FIG. 29C; FIG. 30C). This
result implies that no 2'-3'-cyclic phosphate was formed during the
reaction catalyzed by Case, in contrast to the RuvC-independent
acid-base catalyzed pre-crRNA processing reaction by AsCas12a
(FIGS. 29C and 29D). Together, these data demonstrate that Case
uses a single active site for both pre-crRNA processing and DNA
cleavage, which is a previously unseen activity for a RuvC active
site or a CRISPR-Cas enzyme.
[0727] The versatility and programmability of CRISPR-Cas systems
have sparked a revolution in biotechnology and fundamental
research, as they have been employed to manipulate genomes of
virtually any organism. To investigate whether the DNA cleavage
activity of Cas.PHI. can be harnessed for programmed human genome
editing, a gene disruption assay was performed (Liu et al. (2019)
Nature 566:218; Oakes et al. (2016) Nat. Biotechnol. 34:646) using
Cas.PHI. co-expressed with a suitable crRNA in HEK293 cells (FIG.
32A). It was found that Cas.PHI.-2 and Cas.PHI.-3, but not
Cas.PHI.-1, can induce targeted disruption of a genomically
integrated gene encoding enhanced green fluorescent protein (EGFP)
(FIG. 33A; FIG. 32B). In one case, Case-2 with an individual guide
RNA was able to edit up to 33% of cells (FIG. 33A), comparable to
levels initially reported for CRISPR-Cas9, CRISPR-Cas12a, and
CRISPR-CasX (Zetsche et al. (2015) Cell 163:759; Liu et al. (2019)
supra; Mali et al. (2013) Science 339:823). The small size of
Cas.PHI. in combination with its minimal PAM requirement is
particularly advantageous for both vector-based delivery into cells
and a wider range of targetable genomic sequences, providing a
powerful addition to the CRISPR-Cas toolbox.
[0728] Cas.PHI. represents a new family of CRISPR-Cas enzymes
defined by its single active site for both RNA and DNA cutting.
Three other well-characterized Cas enzymes Cas9, Cas12a, and CasX,
use one (Cas12a and CasX) or two active sites (Cas9) for DNA
cutting and rely on a separate active site (Cas12a) or additional
factors (CasX and Cas9) for crRNA processing (FIG. 33B). The
finding that in Cas.PHI. a single RuvC active site is capable of
both crRNA processing and DNA cutting suggests that size
limitations of phage genomes, possibly in combination with large
population sizes and higher mutation rates in phages compared to
prokaryotes (24-26), led to a consolidation of chemistries within
one catalytic center.
[0729] FIG. 19A-19F. Cas.PHI. is a bona fide CRISPR-Cas system from
huge phages. (A) Maximum Likelihood phylogenetic tree of reported
type V effector proteins and respective predicted ancestral TnpB
nucleases. Bootstrap and approximate likelihood-ratio test values
.gtoreq.90 are denoted on the branches with black circles. (B)
Illustrations of the genomic loci of CRISPR-Cas systems previously
employed in genome editing applications. (C) Graphical
representation of the PAM depletion assay and the resulting PAMs
for three Cas.PHI. orthologs. (D) RNA-sequencing results (left)
mapped onto the native genomic loci of Cas.PHI. orthologs and their
upstream and downstream non-coding regions as cloned into their
respective expression plasmids. Enlarged view of RNA mapped onto
the first repeat-spacer pair (right). (E) Schematic of the
hypothesized function of Biggiephage-encoded Cas.PHI. in an
instance of superinfection of its host. Cas.PHI. may be used by the
huge phage to eliminate competing mobile genetic elements. (F)
Predicted molecular weights of the ribonucleoprotein (RNP)
complexes of small CRISPR-Cas effectors and those functional in
editing of mammalian cells.
[0730] FIG. 20. Maximum likelihood phylogenetic tree of type V
subtypes a-k. Phage-encoded Cas.PHI. proteins are outlined in red,
with prokaryote and transposon-encoded proteins in blue. Bootstrap
and approximate likelihood ratio test values >90 are shown on
the branches (circles).
[0731] FIG. 21. Cas.PHI. crRNA repeats are highly diverse. A
similarity matrix was built and visualized using a heatmap and
hierarchical clustering dendrogram. Cas.PHI.-1, Cas.PHI.-2, and
Cas.PHI.-3 repeats.
[0732] FIG. 22A-22C. Cas.PHI.-3 protects against plasmid
transformation. (A) Scheme illustrating the efficiency of
transformation (EOT) assay. (B) EOT assay showing that Cas.PHI.,
programmed by a beta-lactamase (bla) gene targeting guide, reduces
the efficiency of pUC19 transformation (red bars). Experiments were
performed in three biological replicates and technical
electroporation transformation triplicates (dots; n=3 each,
mean.+-.s.d.). Competent cells were tested for general
transformation efficiency (grey bars) by transformation of pYTK095,
which is not targeted by the tested bla and NT (non-targeting)
guide. (C) EOT in dependence of Cas.PHI.-3 RuvC active site residue
variation (RuvCI: D413A; RuvCII: E618A; RuvCIII: D708A). N=3 each,
mean.+-.s.d.. Competent cells were tested for general
transformation efficiency (grey bars).
[0733] FIG. 23A-23D. Cas.PHI. cleaves DNA. (A) Supercoiled plasmid
cleavage assay in dependence of the guide spacer length. (B)
Cleavage assay targeting dsDNA oligo-duplices for mapping of the
cleavage structure. (C) Scheme illustrating the cleavage pattern.
(D) NTS and TS DNA cleavage efficiency (n=3 each, mean.+-.s.d.).
Data is shown in FIG. 27B.
[0734] FIG. 24A-24D. Purification of apo Cas.PHI.. (A) SDS-PAGE of
the purified apo Cas.PHI. orthologs and their dCas.PHI. variants.
(B) Analytical size-exclusion chromatography (S200) of Cas b-1 WT
(blue trace) and dCas.PHI.-1 (orange trace). (C) Analytical
size-exclusion chromatography (S200) of Cas.PHI.-2 WT (blue trace)
and dCas.PHI.-2 (orange trace). D) Analytical size-exclusion
chromatography (S200) of Cas b-3 WT (blue trace) and dCas.PHI.-3
(orange trace).
[0735] FIG. 25A-25C. Cas.PHI. targets DNA in vitro to produce
staggered cuts. (A) Linear PCR-fragment cleavage assay in
dependence of the guide spacer length and presence of a cognate
5'-TTA-3' PAM (left), or non-cognate 5 `-CCA-3` PAM (right). (B)
Cleavage assay targeting dsDNA oligo-duplices for mapping of the
cleavage structure. (C) Scheme illustrating the cleavage pattern of
the staggered cuts. Shown are the proposed R-loop (replication
loop) structures formed by Cas.PHI., upon target DNA binding to the
crRNA spacer.
[0736] FIG. 26A-26C. Cas.PHI. targets dsDNA and ssDNA, but not RNA
in vitro. (A) Cleavage assay assessing the ability of Cas.PHI., and
dCas.PHI. variant (D371A, D394A and D413A) RNPs to cleave the
target strand (TS), and non-target strand (NTS), of a dsDNA oligo
duplex. (B) Cleavage assay testing the ability of Cas.PHI. and
dCas.PHI. variant (D371A, D394A and D413A) RNPs to target and
cleave a single stranded DNA, or RNA, target strand.
[0737] FIG. 27A-27B. Cleavage assay comparing TS and NTS cleavage
efficiency by Cas.PHI.. (A) Cleavage assay curves, fit to the One
Phase Decay model using Prism 8 (GraphPad) (n=3 each,
mean.+-.s.d.). Cleaved fractions are calculated based on the
substrate band intensities at t=(0 min) (panel B) relative to the
respective time point. (B) Urea-Page gels of the three independent
reaction replicates (Replicates 1, 2 and 3). This panel also
relates to FIG. 23D for Cas.PHI.-2.
[0738] FIG. 28A-28B. Cas.PHI. targets ssDNA, but not RNA, in trans
upon activation in cis. (A) Cleavage assay comparing the trans
cleavage activities of Cas.PHI.-1, Cas.PHI.-2 and Cas.PHI.-3 on
ssDNA and ssRNA as targets in trans in dependence of either ssDNA,
dsDNA, or ssRNA as activators in cis. (B) Cleavage assay comparing
the trans cleavage activity of Cas.PHI.-1, Cas.PHI.-2 and
Cas.PHI.-3.
[0739] FIG. 29A-29D. Cas.PHI. processes pre-crRNA within the RuvC
active site. (A) pre-crRNA substrates and processing sites (red
triangles) as derived from the OH-ladder in panel C. (B) Pre-crRNA
processing assay for Cas.PHI.-1 and Cas.PHI.-2 in dependence of Mg'
and RuvC active site residue variation (D371A and D394A) (n=3 each,
mean.+-.s.d.; t=60 min). Data is shown in FIG. 30B. (C) Left and
middle: Alkaline hydrolysis ladder (OH) of the pre-crRNA substrate.
Right: PNK-phosphatase treatment of the Cas.PHI. and Cas12a
cleavage products. (D) Graphical representation of the mature crRNA
termini chemistry of Cas.PHI. and Cas12a and PNK-phosphorylase
treatment outcomes.
[0740] FIG. 30A-30C. Cas.PHI.-1 and Cas.PHI.-2, but not Cas b-3,
process pre-crRNA. (A) Pre-crRNA processing assay for Cas.PHI.-1,
Cas b-2 and Cas.PHI.-3 in dependence of Mg' and RuvC active site
catalytic residues (dCas.PHI. variants). (A) Processing reaction
replicates for Cas.PHI.-1 and Cas.PHI.-2 at t=0 min and t=60 min.
Purple squares indicate quantified bands. This panel relates to
FIG. 29B. (C) Pre-crRNA processing assay for Cas.PHI.-1, Cas.PHI.-2
and AsCas12a in dependence of Mg' and RuvC active site catalytic
residues (dCas.PHI. variants).
[0741] FIG. 31A-31B. Cas.PHI. WT and dCas.PHI. proteins form RNPs
with pre-crRNA. (A) Analytical size-exclusion chromatography (S200)
of wild-type proteins (blue trace), pre-crRNA (yellow trace), and
their respective reconstituted RNP (green trace). (B) Analytical
size-exclusion chromatography (S200) of dCas.PHI. variant proteins
(blue trace), pre-crRNA (yellow trace), and their respective
reconstituted RNP (green trace).
[0742] FIG. 32A-32C. Cas.PHI. mediated EGFP gene disruption in
HEK293 cells. (A) Schematic of the experimental workflow of the GFP
disruption assay (left) and EGFP disruption by SpyCas9 (right) (B)
Cas.PHI. guides with GFP disruption below 5% (n=3 each,
mean.+-.s.d.). (C) EGFP map showing the target sites and
orientation of guides (arrows and numbers). Yellow triangles
indicate the best guides for gene disruption (relates to FIG. 34A).
Guide sequences are listed in Table 4 (presented in FIG. 35).
[0743] FIG. 33A-33B. Cas.PHI., is functional for human genome
editing. (A) GFP disruption using Cas.PHI.-2 (left) and Cas.PHI.-3
(right) and a non-targeting (NT) guide as a negative control (n=3
each, mean.+-.s.d.). All tested guides and targeted regions within
the EGFP gene are shown in FIG. 32A-32C. (B) Scheme illustrating
the differences in RNA processing and DNA cutting for Cas9, Cas12a,
CasX, and Cas.PHI..
[0744] FIG. 34 presents Table 3.
[0745] FIG. 35 presents Table 4.
[0746] FIG. 36 presents Table 5.
[0747] FIG. 37 presents Table 6.
[0748] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
Sequence CWU 1
1
250136DNAArtificial SequenceSynthetic sequence 1gtctcgacta
atcgagcaat cgtttgagat ctctcc 36237DNAArtificial SequenceSynthetic
sequencemisc_feature(1)..(1)n is a, c, g, or t 2ngtctcgact
aatcgagcaa tcgtttgaga tctctcc 37336DNAArtificial SequenceSynthetic
sequence 3gtcggaacgc tcaacgattg cccctcacga ggggac
36437DNAArtificial SequenceSynthetic sequencemisc_feature(1)..(1)n
is a, c, g, or t 4ngtcggaacg ctcaacgatt gcccctcacg aggggac
37536DNAArtificial SequenceSynthetic sequence 5gtcccagcgt
actgggcaat caatagtcgt tttggt 36637DNAArtificial SequenceSynthetic
sequencemisc_feature(1)..(1)n is a, c, g, or t 6ngtcccagcg
tactgggcaa tcaatagtcg ttttggt 37737DNAArtificial SequenceSynthetic
sequence 7ggatccaatc ctttttgatt gcccaattcg ttgggac
37838DNAArtificial SequenceSynthetic sequencemisc_feature(1)..(1)n
is a, c, g, or t 8nggatccaat cctttttgat tgcccaattc gttgggac
38936DNAArtificial SequenceSynthetic sequence 9ggatctgagg
atcattattg ctcgttacga cgagac 361037DNAArtificial SequenceSynthetic
sequencemisc_feature(1)..(1)n is a, c, g, or t 10nggatctgag
gatcattatt gctcgttacg acgagac 371136DNAArtificial SequenceSynthetic
sequence 11gtctcgtcgt aacgagcaat aatgatcctc agatcc
361237DNAArtificial SequenceSynthetic sequencemisc_feature(1)..(1)n
is a, c, g, or t 12ngtctcgtcg taacgagcaa taatgatcct cagatcc
371336DNAArtificial SequenceSynthetic sequence 13gtctcagcgt
actgagcaat caaaaggttt cgcagg 361437DNAArtificial SequenceSynthetic
sequencemisc_feature(1)..(1)n is a, c, g, or t 14ngtctcagcg
tactgagcaa tcaaaaggtt tcgcagg 371536DNAArtificial SequenceSynthetic
sequence 15gtctcctcgt aaggagcaat ctattagtct tgaaag
361637DNAArtificial SequenceSynthetic sequencemisc_feature(1)..(1)n
is a, c, g, or t 16ngtctcctcg taaggagcaa tctattagtc ttgaaag
371736DNAArtificial SequenceSynthetic sequence 17gtctcggcgc
accgagcaat cagcgaggtc ttctac 361837DNAArtificial SequenceSynthetic
sequencemisc_feature(1)..(1)n is a, c, g, or t 18ngtctcggcg
caccgagcaa tcagcgaggt cttctac 371937DNAArtificial SequenceSynthetic
sequence 19gtcccaacga attgggcaat caaaaaggat tggatcc
372038DNAArtificial SequenceSynthetic sequencemisc_feature(1)..(1)n
is a, c, g, or t 20ngtcccaacg aattgggcaa tcaaaaagga ttggatcc
382136DNAArtificial SequenceSynthetic sequence 21gtcgcggcgt
accgcgcaat gagagtctgt tgccat 362237DNAArtificial SequenceSynthetic
sequencemisc_feature(1)..(1)n is a, c, g, or t 22ngtcgcggcg
taccgcgcaa tgagagtctg ttgccat 372336DNAArtificial SequenceSynthetic
sequence 23accaaaacga ctattgattg cccagtacgc tgggac
362437DNAArtificial SequenceSynthetic sequencemisc_feature(1)..(1)n
is a, c, g, or t 24naccaaaacg actattgatt gcccagtacg ctgggac
372584PRTArtificial SequenceSynthetic sequence 25Met Ala Ser Met
Ile Ser Ser Ser Ala Val Thr Thr Val Ser Arg Ala1 5 10 15Ser Arg Gly
Gln Ser Ala Ala Met Ala Pro Phe Gly Gly Leu Lys Ser 20 25 30Met Thr
Gly Phe Pro Val Arg Lys Val Asn Thr Asp Ile Thr Ser Ile 35 40 45Thr
Ser Asn Gly Gly Arg Val Lys Cys Met Gln Val Trp Pro Pro Ile 50 55
60Gly Lys Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Thr Arg65
70 75 80Asp Ser Arg Ala2657PRTArtificial SequenceSynthetic sequence
26Met Ala Ser Met Ile Ser Ser Ser Ala Val Thr Thr Val Ser Arg Ala1
5 10 15Ser Arg Gly Gln Ser Ala Ala Met Ala Pro Phe Gly Gly Leu Lys
Ser 20 25 30Met Thr Gly Phe Pro Val Arg Lys Val Asn Thr Asp Ile Thr
Ser Ile 35 40 45Thr Ser Asn Gly Gly Arg Val Lys Ser 50
552785PRTArtificial SequenceSynthetic sequence 27Met Ala Ser Ser
Met Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala1 5 10 15Gln Ala Thr
Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala 20 25 30Phe Pro
Ala Thr Arg Lys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser 35 40 45Asn
Gly Gly Arg Val Asn Cys Met Gln Val Trp Pro Pro Ile Glu Lys 50 55
60Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Asp Leu Thr Asp Ser Gly65
70 75 80Gly Arg Val Asn Cys 852876PRTArtificial SequenceSynthetic
sequence 28Met Ala Gln Val Ser Arg Ile Cys Asn Gly Val Gln Asn Pro
Ser Leu1 5 10 15Ile Ser Asn Leu Ser Lys Ser Ser Gln Arg Lys Ser Pro
Leu Ser Val 20 25 30Ser Leu Lys Thr Gln Gln His Pro Arg Ala Tyr Pro
Ile Ser Ser Ser 35 40 45Trp Gly Leu Lys Lys Ser Gly Met Thr Leu Ile
Gly Ser Glu Leu Arg 50 55 60Pro Leu Lys Val Met Ser Ser Val Ser Thr
Ala Cys65 70 752976PRTArtificial SequenceSynthetic sequence 29Met
Ala Gln Val Ser Arg Ile Cys Asn Gly Val Trp Asn Pro Ser Leu1 5 10
15Ile Ser Asn Leu Ser Lys Ser Ser Gln Arg Lys Ser Pro Leu Ser Val
20 25 30Ser Leu Lys Thr Gln Gln His Pro Arg Ala Tyr Pro Ile Ser Ser
Ser 35 40 45Trp Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu
Leu Arg 50 55 60Pro Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys65
70 753072PRTArtificial SequenceSynthetic sequence 30Met Ala Gln Ile
Asn Asn Met Ala Gln Gly Ile Gln Thr Leu Asn Pro1 5 10 15Asn Ser Asn
Phe His Lys Pro Gln Val Pro Lys Ser Ser Ser Phe Leu 20 25 30Val Phe
Gly Ser Lys Lys Leu Lys Asn Ser Ala Asn Ser Met Leu Val 35 40 45Leu
Lys Lys Asp Ser Ile Phe Met Gln Leu Phe Cys Ser Phe Arg Ile 50 55
60Ser Ala Ser Val Ala Thr Ala Cys65 703169PRTArtificial
SequenceSynthetic sequence 31Met Ala Ala Leu Val Thr Ser Gln Leu
Ala Thr Ser Gly Thr Val Leu1 5 10 15Ser Val Thr Asp Arg Phe Arg Arg
Pro Gly Phe Gln Gly Leu Arg Pro 20 25 30Arg Asn Pro Ala Asp Ala Ala
Leu Gly Met Arg Thr Val Gly Ala Ser 35 40 45Ala Ala Pro Lys Gln Ser
Arg Lys Pro His Arg Phe Asp Arg Arg Cys 50 55 60Leu Ser Met Val
Val653277PRTArtificial SequenceSynthetic sequence 32Met Ala Ala Leu
Thr Thr Ser Gln Leu Ala Thr Ser Ala Thr Gly Phe1 5 10 15Gly Ile Ala
Asp Arg Ser Ala Pro Ser Ser Leu Leu Arg His Gly Phe 20 25 30Gln Gly
Leu Lys Pro Arg Ser Pro Ala Gly Gly Asp Ala Thr Ser Leu 35 40 45Ser
Val Thr Thr Ser Ala Arg Ala Thr Pro Lys Gln Gln Arg Ser Val 50 55
60Gln Arg Gly Ser Arg Arg Phe Pro Ser Val Val Val Cys65 70
753357PRTArtificial SequenceSynthetic sequence 33Met Ala Ser Ser
Val Leu Ser Ser Ala Ala Val Ala Thr Arg Ser Asn1 5 10 15Val Ala Gln
Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Ala 20 25 30Ala Ser
Phe Pro Val Ser Arg Lys Gln Asn Leu Asp Ile Thr Ser Ile 35 40 45Ala
Ser Asn Gly Gly Arg Val Gln Cys 50 553465PRTArtificial
SequenceSynthetic sequence 34Met Glu Ser Leu Ala Ala Thr Ser Val
Phe Ala Pro Ser Arg Val Ala1 5 10 15Val Pro Ala Ala Arg Ala Leu Val
Arg Ala Gly Thr Val Val Pro Thr 20 25 30Arg Arg Thr Ser Ser Thr Ser
Gly Thr Ser Gly Val Lys Cys Ser Ala 35 40 45Ala Val Thr Pro Gln Ala
Ser Pro Val Ile Ser Arg Ser Ala Ala Ala 50 55
60Ala653572PRTArtificial SequenceSynthetic sequence 35Met Gly Ala
Ala Ala Thr Ser Met Gln Ser Leu Lys Phe Ser Asn Arg1 5 10 15Leu Val
Pro Pro Ser Arg Arg Leu Ser Pro Val Pro Asn Asn Val Thr 20 25 30Cys
Asn Asn Leu Pro Lys Ser Ala Ala Pro Val Arg Thr Val Lys Cys 35 40
45Cys Ala Ser Ser Trp Asn Ser Thr Ile Asn Gly Ala Ala Ala Thr Thr
50 55 60Asn Gly Ala Ser Ala Ala Ser Ser65 703620PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(4)..(4)The amino acid at
position 4 is selected from lysine, histidine and
arginine.MISC_FEATURE(8)..(8)The amino acid at position 8 is
selected from lysine, histidine and
arginine.MISC_FEATURE(11)..(11)The amino acid at position 11 is
selected from lysine, histidine and
arginine.MISC_FEATURE(15)..(15)The amino acid at position 15 is
selected from lysine, histidine and
arginine.MISC_FEATURE(19)..(19)The amino acid at position 19 is
selected from lysine, histidine and arginine. 36Gly Leu Phe Xaa Ala
Leu Leu Xaa Leu Leu Xaa Ser Leu Trp Xaa Leu1 5 10 15Leu Leu Xaa Ala
203720PRTArtificial SequenceSynthetic sequence 37Gly Leu Phe His
Ala Leu Leu His Leu Leu His Ser Leu Trp His Leu1 5 10 15Leu Leu His
Ala 2038167PRTArtificial SequenceSynthetic sequence 38Met Ser Glu
Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu1 5 10 15Thr Leu
Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30Val
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro 35 40
45Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr
Leu65 70 75 80Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala
Met Ile His 85 90 95Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp
Ala Lys Thr Gly 100 105 110Ala Ala Gly Ser Leu Met Asp Val Leu His
His Pro Gly Met Asn His 115 120 125Arg Val Glu Ile Thr Glu Gly Ile
Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140Leu Ser Asp Phe Phe Arg
Met Arg Arg Gln Glu Ile Lys Ala Gln Lys145 150 155 160Lys Ala Gln
Ser Ser Thr Asp 16539178PRTArtificial SequenceSynthetic sequence
39Met Arg Arg Ala Phe Ile Thr Gly Val Phe Phe Leu Ser Glu Val Glu1
5 10 15Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys
Arg 20 25 30Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu Val
His Asn 35 40 45Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile Gly
Arg His Asp 50 55 60Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
Gly Gly Leu Val65 70 75 80Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr
Leu Tyr Val Thr Leu Glu 85 90 95Pro Cys Val Met Cys Ala Gly Ala Met
Ile His Ser Arg Ile Gly Arg 100 105 110Val Val Phe Gly Ala Arg Asp
Ala Lys Thr Gly Ala Ala Gly Ser Leu 115 120 125Met Asp Val Leu His
His Pro Gly Met Asn His Arg Val Glu Ile Thr 130 135 140Glu Gly Ile
Leu Ala Asp Glu Cys Ala Ala Leu Leu Ser Asp Phe Phe145 150 155
160Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys Ala Gln Ser Ser
165 170 175Thr Asp40160PRTArtificial SequenceSynthetic sequence
40Met Gly Ser His Met Thr Asn Asp Ile Tyr Phe Met Thr Leu Ala Ile1
5 10 15Glu Glu Ala Lys Lys Ala Ala Gln Leu Gly Glu Val Pro Ile Gly
Ala 20 25 30Ile Ile Thr Lys Asp Asp Glu Val Ile Ala Arg Ala His Asn
Leu Arg 35 40 45Glu Thr Leu Gln Gln Pro Thr Ala His Ala Glu His Ile
Ala Ile Glu 50 55 60Arg Ala Ala Lys Val Leu Gly Ser Trp Arg Leu Glu
Gly Cys Thr Leu65 70 75 80Tyr Val Thr Leu Glu Pro Cys Val Met Cys
Ala Gly Thr Ile Val Met 85 90 95Ser Arg Ile Pro Arg Val Val Tyr Gly
Ala Asp Asp Pro Lys Gly Gly 100 105 110Cys Ser Gly Ser Leu Met Asn
Leu Leu Gln Gln Ser Asn Phe Asn His 115 120 125Arg Ala Ile Val Asp
Lys Gly Val Leu Lys Glu Ala Cys Ser Thr Leu 130 135 140Leu Thr Thr
Phe Phe Lys Asn Leu Arg Ala Asn Lys Lys Ser Thr Asn145 150 155
16041161PRTArtificial SequenceSynthetic sequence 41Met Thr Gln Asp
Glu Leu Tyr Met Lys Glu Ala Ile Lys Glu Ala Lys1 5 10 15Lys Ala Glu
Glu Lys Gly Glu Val Pro Ile Gly Ala Val Leu Val Ile 20 25 30Asn Gly
Glu Ile Ile Ala Arg Ala His Asn Leu Arg Glu Thr Glu Gln 35 40 45Arg
Ser Ile Ala His Ala Glu Met Leu Val Ile Asp Glu Ala Cys Lys 50 55
60Ala Leu Gly Thr Trp Arg Leu Glu Gly Ala Thr Leu Tyr Val Thr Leu65
70 75 80Glu Pro Cys Pro Met Cys Ala Gly Ala Val Val Leu Ser Arg Val
Glu 85 90 95Lys Val Val Phe Gly Ala Phe Asp Pro Lys Gly Gly Cys Ser
Gly Thr 100 105 110Leu Met Asn Leu Leu Gln Glu Glu Arg Phe Asn His
Gln Ala Glu Val 115 120 125Val Ser Gly Val Leu Glu Glu Glu Cys Gly
Gly Met Leu Ser Ala Phe 130 135 140Phe Arg Glu Leu Arg Lys Lys Lys
Lys Ala Ala Arg Lys Asn Leu Ser145 150 155 160Glu42183PRTArtificial
SequenceSynthetic sequence 42Met Pro Pro Ala Phe Ile Thr Gly Val
Thr Ser Leu Ser Asp Val Glu1 5 10 15Leu Asp His Glu Tyr Trp Met Arg
His Ala Leu Thr Leu Ala Lys Arg 20 25 30Ala Trp Asp Glu Arg Glu Val
Pro Val Gly Ala Val Leu Val His Asn 35 40 45His Arg Val Ile Gly Glu
Gly Trp Asn Arg Pro Ile Gly Arg His Asp 50 55 60Pro Thr Ala His Ala
Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val65 70 75 80Leu Gln Asn
Tyr Arg Leu Leu Asp Thr Thr Leu Tyr Val Thr Leu Glu 85 90 95Pro Cys
Val Met Cys Ala Gly Ala Met Val His Ser Arg Ile Gly Arg 100 105
110Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala Ala Gly Ser Leu
115 120 125Ile Asp Val Leu His His Pro Gly Met Asn His Arg Val Glu
Ile Ile 130 135 140Glu Gly Val Leu Arg Asp Glu Cys Ala Thr Leu Leu
Ser Asp Phe Phe145 150 155 160Arg Met Arg Arg Gln Glu Ile Lys Ala
Leu Lys Lys Ala Asp Arg Ala 165 170 175Glu Gly Ala Gly Pro Ala Val
18043164PRTArtificial SequenceSynthetic sequence 43Met Asp Glu Tyr
Trp Met Gln Val Ala Met Gln Met Ala Glu Lys Ala1 5 10 15Glu Ala Ala
Gly Glu Val Pro Val Gly Ala Val Leu Val Lys Asp Gly 20 25 30Gln Gln
Ile Ala Thr Gly Tyr Asn Leu Ser Ile Ser Gln His Asp Pro 35 40 45Thr
Ala His Ala Glu Ile Leu Cys Leu Arg Ser Ala Gly Lys Lys Leu 50 55
60Glu Asn Tyr Arg Leu Leu Asp Ala Thr Leu Tyr Ile Thr Leu Glu Pro65
70 75 80Cys Ala Met Cys Ala Gly Ala Met Val His Ser Arg Ile Ala Arg
Val 85 90 95Val Tyr Gly Ala Arg Asp Glu Lys Thr Gly Ala Ala Gly Thr
Val Val 100 105 110Asn Leu Leu Gln His Pro Ala Phe Asn His Gln Val
Glu Val Thr Ser 115 120 125Gly Val Leu Ala Glu Ala Cys Ser Ala Gln
Leu Ser Arg Phe Phe Lys 130 135
140Arg Arg Arg Asp Glu Lys Lys Ala Leu Lys Leu Ala Gln Arg Ala
Gln145 150 155 160Gln Gly Ile Glu44173PRTArtificial
SequenceSynthetic sequence 44Met Asp Ala Ala Lys Val Arg Ser Glu
Phe Asp Glu Lys Met Met Arg1 5 10 15Tyr Ala Leu Glu Leu Ala Asp Lys
Ala Glu Ala Leu Gly Glu Ile Pro 20 25 30Val Gly Ala Val Leu Val Asp
Asp Ala Arg Asn Ile Ile Gly Glu Gly 35 40 45Trp Asn Leu Ser Ile Val
Gln Ser Asp Pro Thr Ala His Ala Glu Ile 50 55 60Ile Ala Leu Arg Asn
Gly Ala Lys Asn Ile Gln Asn Tyr Arg Leu Leu65 70 75 80Asn Ser Thr
Leu Tyr Val Thr Leu Glu Pro Cys Thr Met Cys Ala Gly 85 90 95Ala Ile
Leu His Ser Arg Ile Lys Arg Leu Val Phe Gly Ala Ser Asp 100 105
110Tyr Lys Thr Gly Ala Ile Gly Ser Arg Phe His Phe Phe Asp Asp Tyr
115 120 125Lys Met Asn His Thr Leu Glu Ile Thr Ser Gly Val Leu Ala
Glu Glu 130 135 140Cys Ser Gln Lys Leu Ser Thr Phe Phe Gln Lys Arg
Arg Glu Glu Lys145 150 155 160Lys Ile Glu Lys Ala Leu Leu Lys Ser
Leu Ser Asp Lys 165 17045161PRTArtificial SequenceSynthetic
sequence 45Met Arg Thr Asp Glu Ser Glu Asp Gln Asp His Arg Met Met
Arg Leu1 5 10 15Ala Leu Asp Ala Ala Arg Ala Ala Ala Glu Ala Gly Glu
Thr Pro Val 20 25 30Gly Ala Val Ile Leu Asp Pro Ser Thr Gly Glu Val
Ile Ala Thr Ala 35 40 45Gly Asn Gly Pro Ile Ala Ala His Asp Pro Thr
Ala His Ala Glu Ile 50 55 60Ala Ala Met Arg Ala Ala Ala Ala Lys Leu
Gly Asn Tyr Arg Leu Thr65 70 75 80Asp Leu Thr Leu Val Val Thr Leu
Glu Pro Cys Ala Met Cys Ala Gly 85 90 95Ala Ile Ser His Ala Arg Ile
Gly Arg Val Val Phe Gly Ala Asp Asp 100 105 110Pro Lys Gly Gly Ala
Val Val His Gly Pro Lys Phe Phe Ala Gln Pro 115 120 125Thr Cys His
Trp Arg Pro Glu Val Thr Gly Gly Val Leu Ala Asp Glu 130 135 140Ser
Ala Asp Leu Leu Arg Gly Phe Phe Arg Ala Arg Arg Lys Ala Lys145 150
155 160Ile46179PRTArtificial SequenceSynthetic sequence 46Met Ser
Ser Leu Lys Lys Thr Pro Ile Arg Asp Asp Ala Tyr Trp Met1 5 10 15Gly
Lys Ala Ile Arg Glu Ala Ala Lys Ala Ala Ala Arg Asp Glu Val 20 25
30Pro Ile Gly Ala Val Ile Val Arg Asp Gly Ala Val Ile Gly Arg Gly
35 40 45His Asn Leu Arg Glu Gly Ser Asn Asp Pro Ser Ala His Ala Glu
Met 50 55 60Ile Ala Ile Arg Gln Ala Ala Arg Arg Ser Ala Asn Trp Arg
Leu Thr65 70 75 80Gly Ala Thr Leu Tyr Val Thr Leu Glu Pro Cys Leu
Met Cys Met Gly 85 90 95Ala Ile Ile Leu Ala Arg Leu Glu Arg Val Val
Phe Gly Cys Tyr Asp 100 105 110Pro Lys Gly Gly Ala Ala Gly Ser Leu
Tyr Asp Leu Ser Ala Asp Pro 115 120 125Arg Leu Asn His Gln Val Arg
Leu Ser Pro Gly Val Cys Gln Glu Glu 130 135 140Cys Gly Thr Met Leu
Ser Asp Phe Phe Arg Asp Leu Arg Arg Arg Lys145 150 155 160Lys Ala
Lys Ala Thr Pro Ala Leu Phe Ile Asp Glu Arg Lys Val Pro 165 170
175Pro Glu Pro47198PRTArtificial SequenceSynthetic sequence 47Met
Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys1 5 10
15Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Tyr Val
20 25 30Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser Leu Asp Phe Gly
Tyr 35 40 45Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe Leu
Arg Tyr 50 55 60Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg
Val Thr Trp65 70 75 80Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala
Arg His Val Ala Asp 85 90 95Phe Leu Arg Gly Asn Pro Asn Leu Ser Leu
Arg Ile Phe Thr Ala Arg 100 105 110Leu Tyr Phe Cys Glu Asp Arg Lys
Ala Glu Pro Glu Gly Leu Arg Arg 115 120 125Leu His Arg Ala Gly Val
Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr 130 135 140Phe Tyr Cys Trp
Asn Thr Phe Val Glu Asn His Glu Arg Thr Phe Lys145 150 155 160Ala
Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Arg Gln Leu 165 170
175Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala
180 185 190Phe Arg Thr Leu Gly Leu 19548188PRTArtificial
SequenceSynthetic sequence 48Met Asp Ser Leu Leu Met Asn Arg Arg
Lys Phe Leu Tyr Gln Phe Lys1 5 10 15Asn Val Arg Trp Ala Lys Gly Arg
Arg Glu Thr Tyr Leu Cys Tyr Val 20 25 30Val Lys Arg Arg Asp Ser Ala
Thr Ser Phe Ser Leu Asp Phe Gly Tyr 35 40 45Leu Arg Asn Lys Asn Gly
Cys His Val Glu Leu Leu Phe Leu Arg Tyr 50 55 60Ile Ser Asp Trp Asp
Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp65 70 75 80Phe Thr Ser
Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp 85 90 95Phe Leu
Arg Gly Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg 100 105
110Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg
115 120 125Leu His Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys
Glu Asn 130 135 140His Glu Arg Thr Phe Lys Ala Trp Glu Gly Leu His
Glu Asn Ser Val145 150 155 160Arg Leu Ser Arg Gln Leu Arg Arg Ile
Leu Leu Pro Leu Tyr Glu Val 165 170 175Asp Asp Leu Arg Asp Ala Phe
Arg Thr Leu Gly Leu 180 185497PRTArtificial SequenceSynthetic
sequence 49Pro Lys Lys Lys Arg Lys Val1 55016PRTArtificial
SequenceSynthetic sequence 50Lys Arg Pro Ala Ala Thr Lys Lys Ala
Gly Gln Ala Lys Lys Lys Lys1 5 10 15519PRTArtificial
SequenceSynthetic sequence 51Pro Ala Ala Lys Arg Val Lys Leu Asp1
55211PRTArtificial SequenceSynthetic sequence 52Arg Gln Arg Arg Asn
Glu Leu Lys Arg Ser Pro1 5 105338PRTArtificial SequenceSynthetic
sequence 53Asn Gln Ser Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe
Gly Gly1 5 10 15Arg Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe
Ala Lys Pro 20 25 30Arg Asn Gln Gly Gly Tyr 355442PRTArtificial
SequenceSynthetic sequence 54Arg Met Arg Ile Glx Phe Lys Asn Lys
Gly Lys Asp Thr Ala Glu Leu1 5 10 15Arg Arg Arg Arg Val Glu Val Ser
Val Glu Leu Arg Lys Ala Lys Lys 20 25 30Asp Glu Gln Ile Leu Lys Arg
Arg Asn Val 35 40558PRTArtificial SequenceSynthetic sequence 55Val
Ser Arg Lys Arg Pro Arg Pro1 5568PRTArtificial SequenceSynthetic
sequence 56Pro Gln Pro Lys Lys Lys Pro Leu1 55712PRTArtificial
SequenceSynthetic sequence 57Ser Ala Leu Ile Lys Lys Lys Lys Lys
Met Ala Pro1 5 10585PRTArtificial SequenceSynthetic sequence 58Asp
Arg Leu Arg Arg1 5597PRTArtificial SequenceSynthetic sequence 59Pro
Lys Gln Lys Lys Arg Lys1 56010PRTArtificial SequenceSynthetic
sequence 60Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu1 5
106110PRTArtificial SequenceSynthetic sequence 61Arg Glu Lys Lys
Lys Phe Leu Lys Arg Arg1 5 106220PRTArtificial SequenceSynthetic
sequence 62Lys Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala
Lys Lys1 5 10 15Lys Ser Lys Lys 206317PRTArtificial
SequenceSynthetic sequence 63Arg Lys Cys Leu Gln Ala Gly Met Asn
Leu Glu Ala Arg Lys Thr Lys1 5 10 15Lys6411PRTArtificial
SequenceSynthetic sequence 64Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg1 5 106512PRTArtificial SequenceSynthetic sequence 65Arg Arg
Gln Arg Arg Thr Ser Lys Leu Met Lys Arg1 5 106627PRTArtificial
SequenceSynthetic sequence 66Gly Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Lys Ile Asn Leu1 5 10 15Lys Ala Leu Ala Ala Leu Ala Lys
Lys Ile Leu 20 256733PRTArtificial SequenceSynthetic sequence 67Lys
Ala Leu Ala Trp Glu Ala Lys Leu Ala Lys Ala Leu Ala Lys Ala1 5 10
15Leu Ala Lys His Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Cys Glu
20 25 30Ala6816PRTArtificial SequenceSynthetic sequence 68Arg Gln
Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10
15699PRTArtificial SequenceSynthetic sequence 69Arg Lys Lys Arg Arg
Gln Arg Arg Arg1 5708PRTArtificial SequenceSynthetic sequence 70Arg
Lys Lys Arg Arg Gln Arg Arg1 57111PRTArtificial SequenceSynthetic
sequence 71Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala1 5
107211PRTArtificial SequenceSynthetic sequence 72Thr His Arg Leu
Pro Arg Arg Arg Arg Arg Arg1 5 107311PRTArtificial
SequenceSynthetic sequence 73Gly Gly Arg Arg Ala Arg Arg Arg Arg
Arg Arg1 5 10745PRTArtificial SequenceSynthetic sequence 74Gly Ser
Gly Gly Ser1 5756PRTArtificial SequenceSynthetic sequence 75Gly Gly
Ser Gly Gly Ser1 5764PRTArtificial SequenceSynthetic sequence 76Gly
Gly Gly Ser1774PRTArtificial SequenceSynthetic sequence 77Gly Gly
Ser Gly1785PRTArtificial SequenceSynthetic sequence 78Gly Gly Ser
Gly Gly1 5795PRTArtificial SequenceSynthetic sequence 79Gly Ser Gly
Ser Gly1 5805PRTArtificial SequenceSynthetic sequence 80Gly Ser Gly
Gly Gly1 5815PRTArtificial SequenceSynthetic sequence 81Gly Gly Gly
Ser Gly1 5825PRTArtificial SequenceSynthetic sequence 82Gly Ser Ser
Ser Gly1 58336RNAArtificial SequenceSynthetic sequence 83gucucgacua
aucgagcaau cguuugagau cucucc 368436RNAArtificial SequenceSynthetic
sequence 84gucggaacgc ucaacgauug ccccucacga ggggac
368535RNAArtificial SequenceSynthetic sequence 85gucccagcgu
acugggcaau caauagcguu uuggu 358640RNAArtificial SequenceSynthetic
sequence 86cacaggagag aucucaaacg auugcucgau uagucgagac
408740RNAArtificial SequenceSynthetic sequence 87uaaugucgga
acgcucaacg auugccccuc acgaggggac 408840RNAArtificial
SequenceSynthetic sequence 88auuaaccaaa acgacuauug auugcccagu
acgcugggac 408971RNAArtificial SequenceSynthetic
sequencemisc_feature(1)..(35)n is a, c, g, or u 89nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnngucuc gacuaaucga gcaaucguuu 60gagaucucuc
c 719071RNAArtificial SequenceSynthetic
sequencemisc_feature(1)..(35)n is a, c, g, or u 90nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnngucgg aacgcucaac gauugccccu 60cacgagggga
c 719171RNAArtificial SequenceSynthetic
sequencemisc_feature(37)..(71)n is a, c, g, or u 91gucucgacua
aucgagcaau cguuugagau cucuccnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
n 719271RNAArtificial SequenceSynthetic
sequencemisc_feature(37)..(71)n is a, c, g, or u 92ggagagaucu
caaacgauug cucgauuagu cgagacnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
n 719371RNAArtificial SequenceSynthetic
sequencemisc_feature(37)..(71)n is a, c, g, or u 93gucggaacgc
ucaacgauug ccccucacga ggggacnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
n 719471RNAArtificial SequenceSynthetic
sequencemisc_feature(37)..(71)n is a, c, g, or u 94guccccucgu
gaggggcaau cguugagcgu uccgacnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
n 719575RNAArtificial SequenceSynthetic
sequencemisc_feature(41)..(75)n is a, c, g, or u 95cacaggagag
aucucaaacg auugcucgau uagucgagac nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
nnnnn 759675RNAArtificial SequenceSynthetic
sequencemisc_feature(41)..(75)n is a, c, g, or u 96uaaugucgga
acgcucaacg auugccccuc acgaggggac nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
nnnnn 759775RNAArtificial SequenceSynthetic
sequencemisc_feature(41)..(75)n is a, c, g, or u 97auuaaccaaa
acgacuauug auugcccagu acgcugggac nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
nnnnn 75988PRTArtificial SequenceSynthetic sequence 98Pro Pro Lys
Lys Ala Arg Glu Asp1 59960RNAArtificial SequenceSynthetic sequence
99cacaggagag aucucaaacg auugcucgau uagucgagac agcugguaau gggauaccuu
6010060RNAArtificial SequenceSynthetic sequence 100uaaugucgga
acgcucaacg auugccccuc acgaggggac ugccgccucc gcgacgccca
6010160RNAArtificial SequenceSynthetic sequence 101auuaaccaaa
acgacuauug auugcccagu acgcugggac uaugagcuua uguacaucaa
601021895DNAArtificial SequenceSynthetic sequence 102gctcttgccc
ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 60tcatcattgg
aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
120ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
actttcacca 180gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
aaaaaaggga ataagggcga 240cacggaaatg ttgaatactc atactcttcc
tttttcaata ttattgaagc atttatcagg 300gttattgtct catgagcgga
tacatatttg aatgtattta gaaaaataaa caaatagggg 360ttccgcgcac
atttccccga aaagtgccac ctgtcatgac caaaatccct taacgtgagt
420tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
tgagatcctt 480tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
accgctacca gcggtggttt 540gtttgccgga tcaagagcta ccaactcttt
ttccgaaggt aactggcttc agcagagcgc 600agataccaaa tactgttctt
ctagtgtagc cgtagttagg ccaccacttc aagaactctg 660tagcaccgcc
tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
720ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
gcgcagcggt 780cgggctgaac ggggggttcg tgcacacagc ccagcttgga
gcgaacgacc tacaccgaac 840tgagatacct acagcgtgag ctatgagaaa
gcgccacgct tcccgaaggg agaaaggcgg 900acaggtatcc ggtaagcggc
agggtcggaa caggagagcg cacgagggag cttccagggg 960gaaacgcctg
gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
1020ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
gcggcctttt 1080tacggttcct ggccttttgc tggccttttg ctcacatgtt
ctttcctgcg ttatcccctg 1140attctgtgga taaccgtgcg gccgcccctt
gtagttaagc tggtaatggg ataccttata 1200cagcggccgc gattatcaaa
aaggatcttc acctagatcc ttttaaatta aaaatgaagt 1260tttaaatcaa
tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
1320agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
ctgactcccc 1380gtcgtgtaga taactacgat acgggagggc ttaccatctg
gccccagtgc tgcaatgata 1440ccgcgggacc cacgctcacc ggctccagat
ttatcagcaa taaaccagcc agccggaagg 1500gccgagcgca gaagtggtcc
tgcaacttta tccgcctcca tccagtctat taattgttgc 1560cgggaagcta
gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
1620acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
cggttcccaa 1680cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
aagcggttag ctccttcggt 1740cctccgatcg ttgtcagaag taagttggcc
gcagtgttat cactcatggt tatggcagca 1800ctgcataatt ctcttactgt
catgccatcc gtaagatgct tttctgtgac tggtgagtac 1860tcaaccaagt
cattctgaga atagtgtatg cggcg 18951031895DNAArtificial
SequenceSynthetic sequence 103gctcttgccc ggcgtcaata cgggataata
ccgcgccaca tagcagaact ttaaaagtgc 60tcatcattgg aaaacgttct tcggggcgaa
aactctcaag gatcttaccg ctgttgagat 120ccagttcgat gtaacccact
cgtgcaccca actgatcttc agcatctttt actttcacca 180gcgtttctgg
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
240cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
atttatcagg 300gttattgtct catgagcgga tacatatttg aatgtattta
gaaaaataaa caaatagggg 360ttccgcgcac atttccccga aaagtgccac
ctgtcatgac caaaatccct taacgtgagt 420tttcgttcca ctgagcgtca
gaccccgtag aaaagatcaa aggatcttct tgagatcctt 480tttttctgcg
cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
540gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
agcagagcgc 600agataccaaa tactgttctt ctagtgtagc cgtagttagg
ccaccacttc aagaactctg 660tagcaccgcc
tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
720ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
gcgcagcggt 780cgggctgaac ggggggttcg tgcacacagc ccagcttgga
gcgaacgacc tacaccgaac 840tgagatacct acagcgtgag ctatgagaaa
gcgccacgct tcccgaaggg agaaaggcgg 900acaggtatcc ggtaagcggc
agggtcggaa caggagagcg cacgagggag cttccagggg 960gaaacgcctg
gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
1020ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
gcggcctttt 1080tacggttcct ggccttttgc tggccttttg ctcacatgtt
ctttcctgcg ttatcccctg 1140attctgtgga taaccgtgcg gccgcccctt
gtatttctgc cgcctccgcg acgcccaata 1200cagcggccgc gattatcaaa
aaggatcttc acctagatcc ttttaaatta aaaatgaagt 1260tttaaatcaa
tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
1320agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
ctgactcccc 1380gtcgtgtaga taactacgat acgggagggc ttaccatctg
gccccagtgc tgcaatgata 1440ccgcgggacc cacgctcacc ggctccagat
ttatcagcaa taaaccagcc agccggaagg 1500gccgagcgca gaagtggtcc
tgcaacttta tccgcctcca tccagtctat taattgttgc 1560cgggaagcta
gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
1620acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
cggttcccaa 1680cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
aagcggttag ctccttcggt 1740cctccgatcg ttgtcagaag taagttggcc
gcagtgttat cactcatggt tatggcagca 1800ctgcataatt ctcttactgt
catgccatcc gtaagatgct tttctgtgac tggtgagtac 1860tcaaccaagt
cattctgaga atagtgtatg cggcg 18951041895DNAArtificial
SequenceSynthetic sequence 104gctcttgccc ggcgtcaata cgggataata
ccgcgccaca tagcagaact ttaaaagtgc 60tcatcattgg aaaacgttct tcggggcgaa
aactctcaag gatcttaccg ctgttgagat 120ccagttcgat gtaacccact
cgtgcaccca actgatcttc agcatctttt actttcacca 180gcgtttctgg
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
240cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
atttatcagg 300gttattgtct catgagcgga tacatatttg aatgtattta
gaaaaataaa caaatagggg 360ttccgcgcac atttccccga aaagtgccac
ctgtcatgac caaaatccct taacgtgagt 420tttcgttcca ctgagcgtca
gaccccgtag aaaagatcaa aggatcttct tgagatcctt 480tttttctgcg
cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
540gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
agcagagcgc 600agataccaaa tactgttctt ctagtgtagc cgtagttagg
ccaccacttc aagaactctg 660tagcaccgcc tacatacctc gctctgctaa
tcctgttacc agtggctgct gccagtggcg 720ataagtcgtg tcttaccggg
ttggactcaa gacgatagtt accggataag gcgcagcggt 780cgggctgaac
ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
840tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
agaaaggcgg 900acaggtatcc ggtaagcggc agggtcggaa caggagagcg
cacgagggag cttccagggg 960gaaacgcctg gtatctttat agtcctgtcg
ggtttcgcca cctctgactt gagcgtcgat 1020ttttgtgatg ctcgtcaggg
gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 1080tacggttcct
ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
1140attctgtgga taaccgtgcg gccgcccctt gtaattctat gagcttatgt
acatcaaata 1200cagcggccgc gattatcaaa aaggatcttc acctagatcc
ttttaaatta aaaatgaagt 1260tttaaatcaa tctaaagtat atatgagtaa
acttggtctg acagttacca atgcttaatc 1320agtgaggcac ctatctcagc
gatctgtcta tttcgttcat ccatagttgc ctgactcccc 1380gtcgtgtaga
taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
1440ccgcgggacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
agccggaagg 1500gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
tccagtctat taattgttgc 1560cgggaagcta gagtaagtag ttcgccagtt
aatagtttgc gcaacgttgt tgccattgct 1620acaggcatcg tggtgtcacg
ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 1680cgatcaaggc
gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
1740cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
tatggcagca 1800ctgcataatt ctcttactgt catgccatcc gtaagatgct
tttctgtgac tggtgagtac 1860tcaaccaagt cattctgaga atagtgtatg cggcg
189510520DNAArtificial SequenceSynthetic sequence 105cgtgatggtc
tcgattgagt 2010620DNAArtificial SequenceSynthetic sequence
106accggggtgg tgcccatcct 2010720DNAArtificial SequenceSynthetic
sequence 107atctgcacca ccggcaagct 2010820DNAArtificial
SequenceSynthetic sequence 108gagggcgaca ccctggtgaa
20109707PRTArtificial SequenceSynthetic sequence 109Met Ala Asp Thr
Pro Thr Leu Phe Thr Gln Phe Leu Arg His His Leu1 5 10 15Pro Gly Gln
Arg Phe Arg Lys Asp Ile Leu Lys Gln Ala Gly Arg Ile 20 25 30Leu Ala
Asn Lys Gly Glu Asp Ala Thr Ile Ala Phe Leu Arg Gly Lys 35 40 45Ser
Glu Glu Ser Pro Pro Asp Phe Gln Pro Pro Val Lys Cys Pro Ile 50 55
60Ile Ala Cys Ser Arg Pro Leu Thr Glu Trp Pro Ile Tyr Gln Ala Ser65
70 75 80Val Ala Ile Gln Gly Tyr Val Tyr Gly Gln Ser Leu Ala Glu Phe
Glu 85 90 95Ala Ser Asp Pro Gly Cys Ser Lys Asp Gly Leu Leu Gly Trp
Phe Asp 100 105 110Lys Thr Gly Val Cys Thr Asp Tyr Phe Ser Val Gln
Gly Leu Asn Leu 115 120 125Ile Phe Gln Asn Ala Arg Lys Arg Tyr Ile
Gly Val Gln Thr Lys Val 130 135 140Thr Asn Arg Asn Glu Lys Arg His
Lys Lys Leu Lys Arg Ile Asn Ala145 150 155 160Lys Arg Ile Ala Glu
Gly Leu Pro Glu Leu Thr Ser Asp Glu Pro Glu 165 170 175Ser Ala Leu
Asp Glu Thr Gly His Leu Ile Asp Pro Pro Gly Leu Asn 180 185 190Thr
Asn Ile Tyr Cys Tyr Gln Gln Val Ser Pro Lys Pro Leu Ala Leu 195 200
205Ser Glu Val Asn Gln Leu Pro Thr Ala Tyr Ala Gly Tyr Ser Thr Ser
210 215 220Gly Asp Asp Pro Ile Gln Pro Met Val Thr Lys Asp Arg Leu
Ser Ile225 230 235 240Ser Lys Gly Gln Pro Gly Tyr Ile Pro Glu His
Gln Arg Ala Leu Leu 245 250 255Ser Gln Lys Lys His Arg Arg Met Arg
Gly Tyr Gly Leu Lys Ala Arg 260 265 270Ala Leu Leu Val Ile Val Arg
Ile Gln Asp Asp Trp Ala Val Ile Asp 275 280 285Leu Arg Ser Leu Leu
Arg Asn Ala Tyr Trp Arg Arg Ile Val Gln Thr 290 295 300Lys Glu Pro
Ser Thr Ile Thr Lys Leu Leu Lys Leu Val Thr Gly Asp305 310 315
320Pro Val Leu Asp Ala Thr Arg Met Val Ala Thr Phe Thr Tyr Lys Pro
325 330 335Gly Ile Val Gln Val Arg Ser Ala Lys Cys Leu Lys Asn Lys
Gln Gly 340 345 350Ser Lys Leu Phe Ser Glu Arg Tyr Leu Asn Glu Thr
Val Ser Val Thr 355 360 365Ser Ile Asp Leu Gly Ser Asn Asn Leu Val
Ala Val Ala Thr Tyr Arg 370 375 380Leu Val Asn Gly Asn Thr Pro Glu
Leu Leu Gln Arg Phe Thr Leu Pro385 390 395 400Ser His Leu Val Lys
Asp Phe Glu Arg Tyr Lys Gln Ala His Asp Thr 405 410 415Leu Glu Asp
Ser Ile Gln Lys Thr Ala Val Ala Ser Leu Pro Gln Gly 420 425 430Gln
Gln Thr Glu Ile Arg Met Trp Ser Met Tyr Gly Phe Arg Glu Ala 435 440
445Gln Glu Arg Val Cys Gln Glu Leu Gly Leu Ala Asp Gly Ser Ile Pro
450 455 460Trp Asn Val Met Thr Ala Thr Ser Thr Ile Leu Thr Asp Leu
Phe Leu465 470 475 480Ala Arg Gly Gly Asp Pro Lys Lys Cys Met Phe
Thr Ser Glu Pro Lys 485 490 495Lys Lys Lys Asn Ser Lys Gln Val Leu
Tyr Lys Ile Arg Asp Arg Ala 500 505 510Trp Ala Lys Met Tyr Arg Thr
Leu Leu Ser Lys Glu Thr Arg Glu Ala 515 520 525Trp Asn Lys Ala Leu
Trp Gly Leu Lys Arg Gly Ser Pro Asp Tyr Ala 530 535 540Arg Leu Ser
Lys Arg Lys Glu Glu Leu Ala Arg Arg Cys Val Asn Tyr545 550 555
560Thr Ile Ser Thr Ala Glu Lys Arg Ala Gln Cys Gly Arg Thr Ile Val
565 570 575Ala Leu Glu Asp Leu Asn Ile Gly Phe Phe His Gly Arg Gly
Lys Gln 580 585 590Glu Pro Gly Trp Val Gly Leu Phe Thr Arg Lys Lys
Glu Asn Arg Trp 595 600 605Leu Met Gln Ala Leu His Lys Ala Phe Leu
Glu Leu Ala His His Arg 610 615 620Gly Tyr His Val Ile Glu Val Asn
Pro Ala Tyr Thr Ser Gln Thr Cys625 630 635 640Pro Val Cys Arg His
Cys Asp Pro Asp Asn Arg Asp Gln His Asn Arg 645 650 655Glu Ala Phe
His Cys Ile Gly Cys Gly Phe Arg Gly Asn Ala Asp Leu 660 665 670Asp
Val Ala Thr His Asn Ile Ala Met Val Ala Ile Thr Gly Glu Ser 675 680
685Leu Lys Arg Ala Arg Gly Ser Val Ala Ser Lys Thr Pro Gln Pro Leu
690 695 700Ala Ala Glu705110757PRTArtificial SequenceSynthetic
sequence 110Met Pro Lys Pro Ala Val Glu Ser Glu Phe Ser Lys Val Leu
Lys Lys1 5 10 15His Phe Pro Gly Glu Arg Phe Arg Ser Ser Tyr Met Lys
Arg Gly Gly 20 25 30Lys Ile Leu Ala Ala Gln Gly Glu Glu Ala Val Val
Ala Tyr Leu Gln 35 40 45Gly Lys Ser Glu Glu Glu Pro Pro Asn Phe Gln
Pro Pro Ala Lys Cys 50 55 60His Val Val Thr Lys Ser Arg Asp Phe Ala
Glu Trp Pro Ile Met Lys65 70 75 80Ala Ser Glu Ala Ile Gln Arg Tyr
Ile Tyr Ala Leu Ser Thr Thr Glu 85 90 95Arg Ala Ala Cys Lys Pro Gly
Lys Ser Ser Glu Ser His Ala Ala Trp 100 105 110Phe Ala Ala Thr Gly
Val Ser Asn His Gly Tyr Ser His Val Gln Gly 115 120 125Leu Asn Leu
Ile Phe Asp His Thr Leu Gly Arg Tyr Asp Gly Val Leu 130 135 140Lys
Lys Val Gln Leu Arg Asn Glu Lys Ala Arg Ala Arg Leu Glu Ser145 150
155 160Ile Asn Ala Ser Arg Ala Asp Glu Gly Leu Pro Glu Ile Lys Ala
Glu 165 170 175Glu Glu Glu Val Ala Thr Asn Glu Thr Gly His Leu Leu
Gln Pro Pro 180 185 190Gly Ile Asn Pro Ser Phe Tyr Val Tyr Gln Thr
Ile Ser Pro Gln Ala 195 200 205Tyr Arg Pro Arg Asp Glu Ile Val Leu
Pro Pro Glu Tyr Ala Gly Tyr 210 215 220Val Arg Asp Pro Asn Ala Pro
Ile Pro Leu Gly Val Val Arg Asn Arg225 230 235 240Cys Asp Ile Gln
Lys Gly Cys Pro Gly Tyr Ile Pro Glu Trp Gln Arg 245 250 255Glu Ala
Gly Thr Ala Ile Ser Pro Lys Thr Gly Lys Ala Val Thr Val 260 265
270Pro Gly Leu Ser Pro Lys Lys Asn Lys Arg Met Arg Arg Tyr Trp Arg
275 280 285Ser Glu Lys Glu Lys Ala Gln Asp Ala Leu Leu Val Thr Val
Arg Ile 290 295 300Gly Thr Asp Trp Val Val Ile Asp Val Arg Gly Leu
Leu Arg Asn Ala305 310 315 320Arg Trp Arg Thr Ile Ala Pro Lys Asp
Ile Ser Leu Asn Ala Leu Leu 325 330 335Asp Leu Phe Thr Gly Asp Pro
Val Ile Asp Val Arg Arg Asn Ile Val 340 345 350Thr Phe Thr Tyr Thr
Leu Asp Ala Cys Gly Thr Tyr Ala Arg Lys Trp 355 360 365Thr Leu Lys
Gly Lys Gln Thr Lys Ala Thr Leu Asp Lys Leu Thr Ala 370 375 380Thr
Gln Thr Val Ala Leu Val Ala Ile Asp Leu Gly Gln Thr Asn Pro385 390
395 400Ile Ser Ala Gly Ile Ser Arg Val Thr Gln Glu Asn Gly Ala Leu
Gln 405 410 415Cys Glu Pro Leu Asp Arg Phe Thr Leu Pro Asp Asp Leu
Leu Lys Asp 420 425 430Ile Ser Ala Tyr Arg Ile Ala Trp Asp Arg Asn
Glu Glu Glu Leu Arg 435 440 445Ala Arg Ser Val Glu Ala Leu Pro Glu
Ala Gln Gln Ala Glu Val Arg 450 455 460Ala Leu Asp Gly Val Ser Lys
Glu Thr Ala Arg Thr Gln Leu Cys Ala465 470 475 480Asp Phe Gly Leu
Asp Pro Lys Arg Leu Pro Trp Asp Lys Met Ser Ser 485 490 495Asn Thr
Thr Phe Ile Ser Glu Ala Leu Leu Ser Asn Ser Val Ser Arg 500 505
510Asp Gln Val Phe Phe Thr Pro Ala Pro Lys Lys Gly Ala Lys Lys Lys
515 520 525Ala Pro Val Glu Val Met Arg Lys Asp Arg Thr Trp Ala Arg
Ala Tyr 530 535 540Lys Pro Arg Leu Ser Val Glu Ala Gln Lys Leu Lys
Asn Glu Ala Leu545 550 555 560Trp Ala Leu Lys Arg Thr Ser Pro Glu
Tyr Leu Lys Leu Ser Arg Arg 565 570 575Lys Glu Glu Leu Cys Arg Arg
Ser Ile Asn Tyr Val Ile Glu Lys Thr 580 585 590Arg Arg Arg Thr Gln
Cys Gln Ile Val Ile Pro Val Ile Glu Asp Leu 595 600 605Asn Val Arg
Phe Phe His Gly Ser Gly Lys Arg Leu Pro Gly Trp Asp 610 615 620Asn
Phe Phe Thr Ala Lys Lys Glu Asn Arg Trp Phe Ile Gln Gly Leu625 630
635 640His Lys Ala Phe Ser Asp Leu Arg Thr His Arg Ser Phe Tyr Val
Phe 645 650 655Glu Val Arg Pro Glu Arg Thr Ser Ile Thr Cys Pro Lys
Cys Gly His 660 665 670Cys Glu Val Gly Asn Arg Asp Gly Glu Ala Phe
Gln Cys Leu Ser Cys 675 680 685Gly Lys Thr Cys Asn Ala Asp Leu Asp
Val Ala Thr His Asn Leu Thr 690 695 700Gln Val Ala Leu Thr Gly Lys
Thr Met Pro Lys Arg Glu Glu Pro Arg705 710 715 720Asp Ala Gln Gly
Thr Ala Pro Ala Arg Lys Thr Lys Lys Ala Ser Lys 725 730 735Ser Lys
Ala Pro Pro Ala Glu Arg Glu Asp Gln Thr Pro Ala Gln Glu 740 745
750Pro Ser Gln Thr Ser 755111765PRTArtificial SequenceSynthetic
sequence 111Met Tyr Ile Leu Glu Met Ala Asp Leu Lys Ser Glu Pro Ser
Leu Leu1 5 10 15Ala Lys Leu Leu Arg Asp Arg Phe Pro Gly Lys Tyr Trp
Leu Pro Lys 20 25 30Tyr Trp Lys Leu Ala Glu Lys Lys Arg Leu Thr Gly
Gly Glu Glu Ala 35 40 45Ala Cys Glu Tyr Met Ala Asp Lys Gln Leu Asp
Ser Pro Pro Pro Asn 50 55 60Phe Arg Pro Pro Ala Arg Cys Val Ile Leu
Ala Lys Ser Arg Pro Phe65 70 75 80Glu Asp Trp Pro Val His Arg Val
Ala Ser Lys Ala Gln Ser Phe Val 85 90 95Ile Gly Leu Ser Glu Gln Gly
Phe Ala Ala Leu Arg Ala Ala Pro Pro 100 105 110Ser Thr Ala Asp Ala
Arg Arg Asp Trp Leu Arg Ser His Gly Ala Ser 115 120 125Glu Asp Asp
Leu Met Ala Leu Glu Ala Gln Leu Leu Glu Thr Ile Met 130 135 140Gly
Asn Ala Ile Ser Leu His Gly Gly Val Leu Lys Lys Ile Asp Asn145 150
155 160Ala Asn Val Lys Ala Ala Lys Arg Leu Ser Gly Arg Asn Glu Ala
Arg 165 170 175Leu Asn Lys Gly Leu Gln Glu Leu Pro Pro Glu Gln Glu
Gly Ser Ala 180 185 190Tyr Gly Ala Asp Gly Leu Leu Val Asn Pro Pro
Gly Leu Asn Leu Asn 195 200 205Ile Tyr Cys Arg Lys Ser Cys Cys Pro
Lys Pro Val Lys Asn Thr Ala 210 215 220Arg Phe Val Gly His Tyr Pro
Gly Tyr Leu Arg Asp Ser Asp Ser Ile225 230 235 240Leu Ile Ser Gly
Thr Met Asp Arg Leu Thr Ile Ile Glu Gly Met Pro 245 250 255Gly His
Ile Pro Ala Trp Gln Arg Glu Gln Gly Leu Val Lys Pro Gly 260 265
270Gly Arg Arg Arg Arg Leu Ser Gly Ser Glu Ser Asn Met Arg Gln Lys
275 280 285Val Asp Pro Ser Thr Gly Pro Arg Arg Ser Thr Arg Ser Gly
Thr Val 290 295 300Asn Arg Ser Asn Gln Arg Thr Gly Arg Asn Gly Asp
Pro Leu Leu Val305 310 315 320Glu Ile Arg Met Lys Glu Asp Trp Val
Leu Leu Asp Ala Arg Gly Leu 325 330 335Leu Arg Asn Leu Arg Trp Arg
Glu Ser Lys Arg Gly Leu Ser Cys Asp 340 345 350His Glu Asp Leu Ser
Leu Ser Gly Leu Leu Ala Leu Phe Ser Gly Asp 355 360 365Pro Val Ile
Asp Pro Val Arg Asn Glu Val Val Phe Leu Tyr Gly Glu 370 375 380Gly
Ile Ile Pro Val Arg Ser Thr Lys Pro Val Gly Thr Arg Gln
Ser385 390 395 400Lys Lys Leu Leu Glu Arg Gln Ala Ser Met Gly Pro
Leu Thr Leu Ile 405 410 415Ser Cys Asp Leu Gly Gln Thr Asn Leu Ile
Ala Gly Arg Ala Ser Ala 420 425 430Ile Ser Leu Thr His Gly Ser Leu
Gly Val Arg Ser Ser Val Arg Ile 435 440 445Glu Leu Asp Pro Glu Ile
Ile Lys Ser Phe Glu Arg Leu Arg Lys Asp 450 455 460Ala Asp Arg Leu
Glu Thr Glu Ile Leu Thr Ala Ala Lys Glu Thr Leu465 470 475 480Ser
Asp Glu Gln Arg Gly Glu Val Asn Ser His Glu Lys Asp Ser Pro 485 490
495Gln Thr Ala Lys Ala Ser Leu Cys Arg Glu Leu Gly Leu His Pro Pro
500 505 510Ser Leu Pro Trp Gly Gln Met Gly Pro Ser Thr Thr Phe Ile
Ala Asp 515 520 525Met Leu Ile Ser His Gly Arg Asp Asp Asp Ala Phe
Leu Ser His Gly 530 535 540Glu Phe Pro Thr Leu Glu Lys Arg Lys Lys
Phe Asp Lys Arg Phe Cys545 550 555 560Leu Glu Ser Arg Pro Leu Leu
Ser Ser Glu Thr Arg Lys Ala Leu Asn 565 570 575Glu Ser Leu Trp Glu
Val Lys Arg Thr Ser Ser Glu Tyr Ala Arg Leu 580 585 590Ser Gln Arg
Lys Lys Glu Met Ala Arg Arg Ala Val Asn Phe Val Val 595 600 605Glu
Ile Ser Arg Arg Lys Thr Gly Leu Ser Asn Val Ile Val Asn Ile 610 615
620Glu Asp Leu Asn Val Arg Ile Phe His Gly Gly Gly Lys Gln Ala
Pro625 630 635 640Gly Trp Asp Gly Phe Phe Arg Pro Lys Ser Glu Asn
Arg Trp Phe Ile 645 650 655Gln Ala Ile His Lys Ala Phe Ser Asp Leu
Ala Ala His His Gly Ile 660 665 670Pro Val Ile Glu Ser Asp Pro Gln
Arg Thr Ser Met Thr Cys Pro Glu 675 680 685Cys Gly His Cys Asp Ser
Lys Asn Arg Asn Gly Val Arg Phe Leu Cys 690 695 700Lys Gly Cys Gly
Ala Ser Met Asp Ala Asp Phe Asp Ala Ala Cys Arg705 710 715 720Asn
Leu Glu Arg Val Ala Leu Thr Gly Lys Pro Met Pro Lys Pro Ser 725 730
735Thr Ser Cys Glu Arg Leu Leu Ser Ala Thr Thr Gly Lys Val Cys Ser
740 745 750Asp His Ser Leu Ser His Asp Ala Ile Glu Lys Ala Ser 755
760 765112766PRTArtificial SequenceSynthetic sequence 112Met Glu
Lys Glu Ile Thr Glu Leu Thr Lys Ile Arg Arg Glu Phe Pro1 5 10 15Asn
Lys Lys Phe Ser Ser Thr Asp Met Lys Lys Ala Gly Lys Leu Leu 20 25
30Lys Ala Glu Gly Pro Asp Ala Val Arg Asp Phe Leu Asn Ser Cys Gln
35 40 45Glu Ile Ile Gly Asp Phe Lys Pro Pro Val Lys Thr Asn Ile Val
Ser 50 55 60Ile Ser Arg Pro Phe Glu Glu Trp Pro Val Ser Met Val Gly
Arg Ala65 70 75 80Ile Gln Glu Tyr Tyr Phe Ser Leu Thr Lys Glu Glu
Leu Glu Ser Val 85 90 95His Pro Gly Thr Ser Ser Glu Asp His Lys Ser
Phe Phe Asn Ile Thr 100 105 110Gly Leu Ser Asn Tyr Asn Tyr Thr Ser
Val Gln Gly Leu Asn Leu Ile 115 120 125Phe Lys Asn Ala Lys Ala Ile
Tyr Asp Gly Thr Leu Val Lys Ala Asn 130 135 140Asn Lys Asn Lys Lys
Leu Glu Lys Lys Phe Asn Glu Ile Asn His Lys145 150 155 160Arg Ser
Leu Glu Gly Leu Pro Ile Ile Thr Pro Asp Phe Glu Glu Pro 165 170
175Phe Asp Glu Asn Gly His Leu Asn Asn Pro Pro Gly Ile Asn Arg Asn
180 185 190Ile Tyr Gly Tyr Gln Gly Cys Ala Ala Lys Val Phe Val Pro
Ser Lys 195 200 205His Lys Met Val Ser Leu Pro Lys Glu Tyr Glu Gly
Tyr Asn Arg Asp 210 215 220Pro Asn Leu Ser Leu Ala Gly Phe Arg Asn
Arg Leu Glu Ile Pro Glu225 230 235 240Gly Glu Pro Gly His Val Pro
Trp Phe Gln Arg Met Asp Ile Pro Glu 245 250 255Gly Gln Ile Gly His
Val Asn Lys Ile Gln Arg Phe Asn Phe Val His 260 265 270Gly Lys Asn
Ser Gly Lys Val Lys Phe Ser Asp Lys Thr Gly Arg Val 275 280 285Lys
Arg Tyr His His Ser Lys Tyr Lys Asp Ala Thr Lys Pro Tyr Lys 290 295
300Phe Leu Glu Glu Ser Lys Lys Val Ser Ala Leu Asp Ser Ile Leu
Ala305 310 315 320Ile Ile Thr Ile Gly Asp Asp Trp Val Val Phe Asp
Ile Arg Gly Leu 325 330 335Tyr Arg Asn Val Phe Tyr Arg Glu Leu Ala
Gln Lys Gly Leu Thr Ala 340 345 350Val Gln Leu Leu Asp Leu Phe Thr
Gly Asp Pro Val Ile Asp Pro Lys 355 360 365Lys Gly Val Val Thr Phe
Ser Tyr Lys Glu Gly Val Val Pro Val Phe 370 375 380Ser Gln Lys Ile
Val Pro Arg Phe Lys Ser Arg Asp Thr Leu Glu Lys385 390 395 400Leu
Thr Ser Gln Gly Pro Val Ala Leu Leu Ser Val Asp Leu Gly Gln 405 410
415Asn Glu Pro Val Ala Ala Arg Val Cys Ser Leu Lys Asn Ile Asn Asp
420 425 430Lys Ile Thr Leu Asp Asn Ser Cys Arg Ile Ser Phe Leu Asp
Asp Tyr 435 440 445Lys Lys Gln Ile Lys Asp Tyr Arg Asp Ser Leu Asp
Glu Leu Glu Ile 450 455 460Lys Ile Arg Leu Glu Ala Ile Asn Ser Leu
Glu Thr Asn Gln Gln Val465 470 475 480Glu Ile Arg Asp Leu Asp Val
Phe Ser Ala Asp Arg Ala Lys Ala Asn 485 490 495Thr Val Asp Met Phe
Asp Ile Asp Pro Asn Leu Ile Ser Trp Asp Ser 500 505 510Met Ser Asp
Ala Arg Val Ser Thr Gln Ile Ser Asp Leu Tyr Leu Lys 515 520 525Asn
Gly Gly Asp Glu Ser Arg Val Tyr Phe Glu Ile Asn Asn Lys Arg 530 535
540Ile Lys Arg Ser Asp Tyr Asn Ile Ser Gln Leu Val Arg Pro Lys
Leu545 550 555 560Ser Asp Ser Thr Arg Lys Asn Leu Asn Asp Ser Ile
Trp Lys Leu Lys 565 570 575Arg Thr Ser Glu Glu Tyr Leu Lys Leu Ser
Lys Arg Lys Leu Glu Leu 580 585 590Ser Arg Ala Val Val Asn Tyr Thr
Ile Arg Gln Ser Lys Leu Leu Ser 595 600 605Gly Ile Asn Asp Ile Val
Ile Ile Leu Glu Asp Leu Asp Val Lys Lys 610 615 620Lys Phe Asn Gly
Arg Gly Ile Arg Asp Ile Gly Trp Asp Asn Phe Phe625 630 635 640Ser
Ser Arg Lys Glu Asn Arg Trp Phe Ile Pro Ala Phe His Lys Ala 645 650
655Phe Ser Glu Leu Ser Ser Asn Arg Gly Leu Cys Val Ile Glu Val Asn
660 665 670Pro Ala Trp Thr Ser Ala Thr Cys Pro Asp Cys Gly Phe Cys
Ser Lys 675 680 685Glu Asn Arg Asp Gly Ile Asn Phe Thr Cys Arg Lys
Cys Gly Val Ser 690 695 700Tyr His Ala Asp Ile Asp Val Ala Thr Leu
Asn Ile Ala Arg Val Ala705 710 715 720Val Leu Gly Lys Pro Met Ser
Gly Pro Ala Asp Arg Glu Arg Leu Gly 725 730 735Asp Thr Lys Lys Pro
Arg Val Ala Arg Ser Arg Lys Thr Met Lys Arg 740 745 750Lys Asp Ile
Ser Asn Ser Thr Val Glu Ala Met Val Thr Ala 755 760
765113812PRTArtificial SequenceSynthetic sequence 113Met Asp Met
Leu Asp Thr Glu Thr Asn Tyr Ala Thr Glu Thr Pro Ala1 5 10 15Gln Gln
Gln Asp Tyr Ser Pro Lys Pro Pro Lys Lys Ala Gln Arg Ala 20 25 30Pro
Lys Gly Phe Ser Lys Lys Ala Arg Pro Glu Lys Lys Pro Pro Lys 35 40
45Pro Ile Thr Leu Phe Thr Gln Lys His Phe Ser Gly Val Arg Phe Leu
50 55 60Lys Arg Val Ile Arg Asp Ala Ser Lys Ile Leu Lys Leu Ser Glu
Ser65 70 75 80Arg Thr Ile Thr Phe Leu Glu Gln Ala Ile Glu Arg Asp
Gly Ser Ala 85 90 95Pro Pro Asp Val Thr Pro Pro Val His Asn Thr Ile
Met Ala Val Thr 100 105 110Arg Pro Phe Glu Glu Trp Pro Glu Val Ile
Leu Ser Lys Ala Leu Gln 115 120 125Lys His Cys Tyr Ala Leu Thr Lys
Lys Ile Lys Ile Lys Thr Trp Pro 130 135 140Lys Lys Gly Pro Gly Lys
Lys Cys Leu Ala Ala Trp Ser Ala Arg Thr145 150 155 160Lys Ile Pro
Leu Ile Pro Gly Gln Val Gln Ala Thr Asn Gly Leu Phe 165 170 175Asp
Arg Ile Gly Ser Ile Tyr Asp Gly Val Glu Lys Lys Val Thr Asn 180 185
190Arg Asn Ala Asn Lys Lys Leu Glu Tyr Asp Glu Ala Ile Lys Glu Gly
195 200 205Arg Asn Pro Ala Val Pro Glu Tyr Glu Thr Ala Tyr Asn Ile
Asp Gly 210 215 220Thr Leu Ile Asn Lys Pro Gly Tyr Asn Pro Asn Leu
Tyr Ile Thr Gln225 230 235 240Ser Arg Thr Pro Arg Leu Ile Thr Glu
Ala Asp Arg Pro Leu Val Glu 245 250 255Lys Ile Leu Trp Gln Met Val
Glu Lys Lys Thr Gln Ser Arg Asn Gln 260 265 270Ala Arg Arg Ala Arg
Leu Glu Lys Ala Ala His Leu Gln Gly Leu Pro 275 280 285Val Pro Lys
Phe Val Pro Glu Lys Val Asp Arg Ser Gln Lys Ile Glu 290 295 300Ile
Arg Ile Ile Asp Pro Leu Asp Lys Ile Glu Pro Tyr Met Pro Gln305 310
315 320Asp Arg Met Ala Ile Lys Ala Ser Gln Asp Gly His Val Pro Tyr
Trp 325 330 335Gln Arg Pro Phe Leu Ser Lys Arg Arg Asn Arg Arg Val
Arg Ala Gly 340 345 350Trp Gly Lys Gln Val Ser Ser Ile Gln Ala Trp
Leu Thr Gly Ala Leu 355 360 365Leu Val Ile Val Arg Leu Gly Asn Glu
Ala Phe Leu Ala Asp Ile Arg 370 375 380Gly Ala Leu Arg Asn Ala Gln
Trp Arg Lys Leu Leu Lys Pro Asp Ala385 390 395 400Thr Tyr Gln Ser
Leu Phe Asn Leu Phe Thr Gly Asp Pro Val Val Asn 405 410 415Thr Arg
Thr Asn His Leu Thr Met Ala Tyr Arg Glu Gly Val Val Asn 420 425
430Ile Val Lys Ser Arg Ser Phe Lys Gly Arg Gln Thr Arg Glu His Leu
435 440 445Leu Thr Leu Leu Gly Gln Gly Lys Thr Val Ala Gly Val Ser
Phe Asp 450 455 460Leu Gly Gln Lys His Ala Ala Gly Leu Leu Ala Ala
His Phe Gly Leu465 470 475 480Gly Glu Asp Gly Asn Pro Val Phe Thr
Pro Ile Gln Ala Cys Phe Leu 485 490 495Pro Gln Arg Tyr Leu Asp Ser
Leu Thr Asn Tyr Arg Asn Arg Tyr Asp 500 505 510Ala Leu Thr Leu Asp
Met Arg Arg Gln Ser Leu Leu Ala Leu Thr Pro 515 520 525Ala Gln Gln
Gln Glu Phe Ala Asp Ala Gln Arg Asp Pro Gly Gly Gln 530 535 540Ala
Lys Arg Ala Cys Cys Leu Lys Leu Asn Leu Asn Pro Asp Glu Ile545 550
555 560Arg Trp Asp Leu Val Ser Gly Ile Ser Thr Met Ile Ser Asp Leu
Tyr 565 570 575Ile Glu Arg Gly Gly Asp Pro Arg Asp Val His Gln Gln
Val Glu Thr 580 585 590Lys Pro Lys Gly Lys Arg Lys Ser Glu Ile Arg
Ile Leu Lys Ile Arg 595 600 605Asp Gly Lys Trp Ala Tyr Asp Phe Arg
Pro Lys Ile Ala Asp Glu Thr 610 615 620Arg Lys Ala Gln Arg Glu Gln
Leu Trp Lys Leu Gln Lys Ala Ser Ser625 630 635 640Glu Phe Glu Arg
Leu Ser Arg Tyr Lys Ile Asn Ile Ala Arg Ala Ile 645 650 655Ala Asn
Trp Ala Leu Gln Trp Gly Arg Glu Leu Ser Gly Cys Asp Ile 660 665
670Val Ile Pro Val Leu Glu Asp Leu Asn Val Gly Ser Lys Phe Phe Asp
675 680 685Gly Lys Gly Lys Trp Leu Leu Gly Trp Asp Asn Arg Phe Thr
Pro Lys 690 695 700Lys Glu Asn Arg Trp Phe Ile Lys Val Leu His Lys
Ala Val Ala Glu705 710 715 720Leu Ala Pro His Arg Gly Val Pro Val
Tyr Glu Val Met Pro His Arg 725 730 735Thr Ser Met Thr Cys Pro Ala
Cys His Tyr Cys His Pro Thr Asn Arg 740 745 750Glu Gly Asp Arg Phe
Glu Cys Gln Ser Cys His Val Val Lys Asn Thr 755 760 765Asp Arg Asp
Val Ala Pro Tyr Asn Ile Leu Arg Val Ala Val Glu Gly 770 775 780Lys
Thr Leu Asp Arg Trp Gln Ala Glu Lys Lys Pro Gln Ala Glu Pro785 790
795 800Asp Arg Pro Met Ile Leu Ile Asp Asn Gln Glu Ser 805
810114812PRTArtificial SequenceSynthetic sequence 114Met Asp Met
Leu Asp Thr Glu Thr Asn Tyr Ala Thr Glu Thr Pro Ala1 5 10 15Gln Gln
Gln Asp Tyr Ser Pro Lys Pro Pro Lys Lys Ala Gln Arg Ala 20 25 30Pro
Lys Gly Phe Ser Lys Lys Ala Arg Pro Glu Lys Lys Pro Pro Lys 35 40
45Pro Ile Thr Leu Phe Thr Gln Lys His Phe Ser Gly Val Arg Phe Leu
50 55 60Lys Arg Val Ile Arg Asp Ala Ser Lys Ile Leu Lys Leu Ser Glu
Ser65 70 75 80Arg Thr Ile Thr Phe Leu Glu Gln Ala Ile Glu Arg Asp
Gly Ser Ala 85 90 95Pro Pro Asp Val Thr Pro Pro Val His Asn Thr Ile
Met Ala Val Thr 100 105 110Arg Pro Phe Glu Glu Trp Pro Glu Val Ile
Leu Ser Lys Ala Leu Gln 115 120 125Lys His Cys Tyr Ala Leu Thr Lys
Lys Ile Lys Ile Lys Thr Trp Pro 130 135 140Lys Lys Gly Pro Gly Lys
Lys Cys Leu Ala Ala Trp Ser Ala Arg Thr145 150 155 160Lys Ile Pro
Leu Ile Pro Gly Gln Val Gln Ala Thr Asn Gly Leu Phe 165 170 175Asp
Arg Ile Gly Ser Ile Tyr Asp Gly Val Glu Lys Lys Val Thr Asn 180 185
190Arg Asn Ala Asn Lys Lys Leu Glu Tyr Asp Glu Ala Ile Lys Glu Gly
195 200 205Arg Asn Pro Ala Val Pro Glu Tyr Glu Thr Ala Tyr Asn Ile
Asp Gly 210 215 220Thr Leu Ile Asn Lys Pro Gly Tyr Asn Pro Asn Leu
Tyr Ile Thr Gln225 230 235 240Ser Arg Thr Pro Arg Leu Ile Thr Glu
Ala Asp Arg Pro Leu Val Glu 245 250 255Lys Ile Leu Trp Gln Met Val
Glu Lys Lys Thr Gln Ser Arg Asn Gln 260 265 270Ala Arg Arg Ala Arg
Leu Glu Lys Ala Ala His Leu Gln Gly Leu Pro 275 280 285Val Pro Lys
Phe Val Pro Glu Lys Val Asp Arg Ser Gln Lys Ile Glu 290 295 300Ile
Arg Ile Ile Asp Pro Leu Asp Lys Ile Glu Pro Tyr Met Pro Gln305 310
315 320Asp Arg Met Ala Ile Lys Ala Ser Gln Asp Gly His Val Pro Tyr
Trp 325 330 335Gln Arg Pro Phe Leu Ser Lys Arg Arg Asn Arg Arg Val
Arg Ala Gly 340 345 350Trp Gly Lys Gln Val Ser Ser Ile Gln Ala Trp
Leu Thr Gly Ala Leu 355 360 365Leu Val Ile Val Arg Leu Gly Asn Glu
Ala Phe Leu Ala Asp Ile Arg 370 375 380Gly Ala Leu Arg Asn Ala Gln
Trp Arg Lys Leu Leu Lys Pro Asp Ala385 390 395 400Thr Tyr Gln Ser
Leu Phe Asn Leu Phe Thr Gly Asp Pro Val Val Asn 405 410 415Thr Arg
Thr Asn His Leu Thr Met Ala Tyr Arg Glu Gly Val Val Asp 420 425
430Ile Val Lys Ser Arg Ser Phe Lys Gly Arg Gln Thr Arg Glu His Leu
435 440 445Leu Thr Leu Leu Gly Gln Gly Lys Thr Val Ala Gly Val Ser
Phe Asp 450 455 460Leu Gly Gln Lys His Ala Ala Gly Leu Leu Ala Ala
His Phe Gly Leu465 470 475 480Gly Glu Asp Gly Asn Pro Val Phe Thr
Pro Ile Gln Ala Cys Phe Leu 485 490 495Pro Gln Arg Tyr Leu Asp
Ser
Leu Thr Asn Tyr Arg Asn Arg Tyr Asp 500 505 510Ala Leu Thr Leu Asp
Met Arg Arg Gln Ser Leu Leu Ala Leu Thr Pro 515 520 525Ala Gln Gln
Gln Glu Phe Ala Asp Ala Gln Arg Asp Pro Gly Gly Gln 530 535 540Ala
Lys Arg Ala Cys Cys Leu Lys Leu Asn Leu Asn Pro Asp Glu Ile545 550
555 560Arg Trp Asp Leu Val Ser Gly Ile Ser Thr Met Ile Ser Asp Leu
Tyr 565 570 575Ile Glu Arg Gly Gly Asp Pro Arg Asp Val His Gln Gln
Val Glu Thr 580 585 590Lys Pro Lys Gly Lys Arg Lys Ser Glu Ile Arg
Ile Leu Lys Ile Arg 595 600 605Asp Gly Lys Trp Ala Tyr Asp Phe Arg
Pro Lys Ile Ala Asp Glu Thr 610 615 620Arg Lys Ala Gln Arg Glu Gln
Leu Trp Lys Leu Gln Lys Ala Ser Ser625 630 635 640Glu Phe Glu Arg
Leu Ser Arg Tyr Lys Ile Asn Ile Ala Arg Ala Ile 645 650 655Ala Asn
Trp Ala Leu Gln Trp Gly Arg Glu Leu Ser Gly Cys Asp Ile 660 665
670Val Ile Pro Val Leu Glu Asp Leu Asn Val Gly Ser Lys Phe Phe Asp
675 680 685Gly Lys Gly Lys Trp Leu Leu Gly Trp Asp Asn Arg Phe Thr
Pro Lys 690 695 700Lys Glu Asn Arg Trp Phe Ile Lys Val Leu His Lys
Ala Val Ala Glu705 710 715 720Leu Ala Pro His Lys Gly Val Pro Val
Tyr Glu Val Met Pro His Arg 725 730 735Thr Ser Met Thr Cys Pro Ala
Cys His Tyr Cys His Pro Thr Asn Arg 740 745 750Glu Gly Asp Arg Phe
Glu Cys Gln Ser Cys His Val Val Lys Asn Thr 755 760 765Asp Arg Asp
Val Ala Pro Tyr Asn Ile Leu Arg Val Ala Val Glu Gly 770 775 780Lys
Thr Leu Asp Arg Trp Gln Ala Glu Lys Lys Pro Gln Ala Glu Pro785 790
795 800Asp Arg Pro Met Ile Leu Ile Asp Asn Gln Glu Ser 805
810115793PRTArtificial SequenceSynthetic sequence 115Met Ser Ser
Leu Pro Thr Pro Leu Glu Leu Leu Lys Gln Lys His Ala1 5 10 15Asp Leu
Phe Lys Gly Leu Gln Phe Ser Ser Lys Asp Asn Lys Met Ala 20 25 30Gly
Lys Val Leu Lys Lys Asp Gly Glu Glu Ala Ala Leu Ala Phe Leu 35 40
45Ser Glu Arg Gly Val Ser Arg Gly Glu Leu Pro Asn Phe Arg Pro Pro
50 55 60Ala Lys Thr Leu Val Val Ala Gln Ser Arg Pro Phe Glu Glu Phe
Pro65 70 75 80Ile Tyr Arg Val Ser Glu Ala Ile Gln Leu Tyr Val Tyr
Ser Leu Ser 85 90 95Val Lys Glu Leu Glu Thr Val Pro Ser Gly Ser Ser
Thr Lys Lys Glu 100 105 110His Gln Arg Phe Phe Gln Asp Ser Ser Val
Pro Asp Phe Gly Tyr Thr 115 120 125Ser Val Gln Gly Leu Asn Lys Ile
Phe Gly Leu Ala Arg Gly Ile Tyr 130 135 140Leu Gly Val Ile Thr Arg
Gly Glu Asn Gln Leu Gln Lys Ala Lys Ser145 150 155 160Lys His Glu
Ala Leu Asn Lys Lys Arg Arg Ala Ser Gly Glu Ala Glu 165 170 175Thr
Glu Phe Asp Pro Thr Pro Tyr Glu Tyr Met Thr Pro Glu Arg Lys 180 185
190Leu Ala Lys Pro Pro Gly Val Asn His Ser Ile Met Cys Tyr Val Asp
195 200 205Ile Ser Val Asp Glu Phe Asp Phe Arg Asn Pro Asp Gly Ile
Val Leu 210 215 220Pro Ser Glu Tyr Ala Gly Tyr Cys Arg Glu Ile Asn
Thr Ala Ile Glu225 230 235 240Lys Gly Thr Val Asp Arg Leu Gly His
Leu Lys Gly Gly Pro Gly Tyr 245 250 255Ile Pro Gly His Gln Arg Lys
Glu Ser Thr Thr Glu Gly Pro Lys Ile 260 265 270Asn Phe Arg Lys Gly
Arg Ile Arg Arg Ser Tyr Thr Ala Leu Tyr Ala 275 280 285Lys Arg Asp
Ser Arg Arg Val Arg Gln Gly Lys Leu Ala Leu Pro Ser 290 295 300Tyr
Arg His His Met Met Arg Leu Asn Ser Asn Ala Glu Ser Ala Ile305 310
315 320Leu Ala Val Ile Phe Phe Gly Lys Asp Trp Val Val Phe Asp Leu
Arg 325 330 335Gly Leu Leu Arg Asn Val Arg Trp Arg Asn Leu Phe Val
Asp Gly Ser 340 345 350Thr Pro Ser Thr Leu Leu Gly Met Phe Gly Asp
Pro Val Ile Asp Pro 355 360 365Lys Arg Gly Val Val Ala Phe Cys Tyr
Lys Glu Gln Ile Val Pro Val 370 375 380Val Ser Lys Ser Ile Thr Lys
Met Val Lys Ala Pro Glu Leu Leu Asn385 390 395 400Lys Leu Tyr Leu
Lys Ser Glu Asp Pro Leu Val Leu Val Ala Ile Asp 405 410 415Leu Gly
Gln Thr Asn Pro Val Gly Val Gly Val Tyr Arg Val Met Asn 420 425
430Ala Ser Leu Asp Tyr Glu Val Val Thr Arg Phe Ala Leu Glu Ser Glu
435 440 445Leu Leu Arg Glu Ile Glu Ser Tyr Arg Gln Arg Thr Asn Ala
Phe Glu 450 455 460Ala Gln Ile Arg Ala Glu Thr Phe Asp Ala Met Thr
Ser Glu Glu Gln465 470 475 480Glu Glu Ile Thr Arg Val Arg Ala Phe
Ser Ala Ser Lys Ala Lys Glu 485 490 495Asn Val Cys His Arg Phe Gly
Met Pro Val Asp Ala Val Asp Trp Ala 500 505 510Thr Met Gly Ser Asn
Thr Ile His Ile Ala Lys Trp Val Met Arg His 515 520 525Gly Asp Pro
Ser Leu Val Glu Val Leu Glu Tyr Arg Lys Asp Asn Glu 530 535 540Ile
Lys Leu Asp Lys Asn Gly Val Pro Lys Lys Val Lys Leu Thr Asp545 550
555 560Lys Arg Ile Ala Asn Leu Thr Ser Ile Arg Leu Arg Phe Ser Gln
Glu 565 570 575Thr Ser Lys His Tyr Asn Asp Thr Met Trp Glu Leu Arg
Arg Lys His 580 585 590Pro Val Tyr Gln Lys Leu Ser Lys Ser Lys Ala
Asp Phe Ser Arg Arg 595 600 605Val Val Asn Ser Ile Ile Arg Arg Val
Asn His Leu Val Pro Arg Ala 610 615 620Arg Ile Val Phe Ile Ile Glu
Asp Leu Lys Asn Leu Gly Lys Val Phe625 630 635 640His Gly Ser Gly
Lys Arg Glu Leu Gly Trp Asp Ser Tyr Phe Glu Pro 645 650 655Lys Ser
Glu Asn Arg Trp Phe Ile Gln Val Leu His Lys Ala Phe Ser 660 665
670Glu Thr Gly Lys His Lys Gly Tyr Tyr Ile Ile Glu Cys Trp Pro Asn
675 680 685Trp Thr Ser Cys Thr Cys Pro Lys Cys Ser Cys Cys Asp Ser
Glu Asn 690 695 700Arg His Gly Glu Val Phe Arg Cys Leu Ala Cys Gly
Tyr Thr Cys Asn705 710 715 720Thr Asp Phe Gly Thr Ala Pro Asp Asn
Leu Val Lys Ile Ala Thr Thr 725 730 735Gly Lys Gly Leu Pro Gly Pro
Lys Lys Arg Cys Lys Gly Ser Ser Lys 740 745 750Gly Lys Asn Pro Lys
Ile Ala Arg Ser Ser Glu Thr Gly Val Ser Val 755 760 765Thr Glu Ser
Gly Ala Pro Lys Val Lys Lys Ser Ser Pro Thr Gln Thr 770 775 780Ser
Gln Ser Ser Ser Gln Ser Ala Pro785 790116441PRTArtificial
SequenceSynthetic sequence 116Met Asn Lys Ile Glu Lys Glu Lys Thr
Pro Leu Ala Lys Leu Met Asn1 5 10 15Glu Asn Phe Ala Gly Leu Arg Phe
Pro Phe Ala Ile Ile Lys Gln Ala 20 25 30Gly Lys Lys Leu Leu Lys Glu
Gly Glu Leu Lys Thr Ile Glu Tyr Met 35 40 45Thr Gly Lys Gly Ser Ile
Glu Pro Leu Pro Asn Phe Lys Pro Pro Val 50 55 60Lys Cys Leu Ile Val
Ala Lys Arg Arg Asp Leu Lys Tyr Phe Pro Ile65 70 75 80Cys Lys Ala
Ser Cys Glu Ile Gln Ser Tyr Val Tyr Ser Leu Asn Tyr 85 90 95Lys Asp
Phe Met Asp Tyr Phe Ser Thr Pro Met Thr Ser Gln Lys Gln 100 105
110His Glu Glu Phe Phe Lys Lys Ser Gly Leu Asn Ile Glu Tyr Gln Asn
115 120 125Val Ala Gly Leu Asn Leu Ile Phe Asn Asn Val Lys Asn Thr
Tyr Asn 130 135 140Gly Val Ile Leu Lys Val Lys Asn Arg Asn Glu Lys
Leu Lys Lys Lys145 150 155 160Ala Ile Lys Asn Asn Tyr Glu Phe Glu
Glu Ile Lys Thr Phe Asn Asp 165 170 175Asp Gly Cys Leu Ile Asn Lys
Pro Gly Ile Asn Asn Val Ile Tyr Cys 180 185 190Phe Gln Ser Ile Ser
Pro Lys Ile Leu Lys Asn Ile Thr His Leu Pro 195 200 205Lys Glu Tyr
Asn Asp Tyr Asp Cys Ser Val Asp Arg Asn Ile Ile Gln 210 215 220Lys
Tyr Val Ser Arg Leu Asp Ile Pro Glu Ser Gln Pro Gly His Val225 230
235 240Pro Glu Trp Gln Arg Lys Leu Pro Glu Phe Asn Asn Thr Asn Asn
Pro 245 250 255Arg Arg Arg Arg Lys Trp Tyr Ser Asn Gly Arg Asn Ile
Ser Lys Gly 260 265 270Tyr Ser Val Asp Gln Val Asn Gln Ala Lys Ile
Glu Asp Ser Leu Leu 275 280 285Ala Gln Ile Lys Ile Gly Glu Asp Trp
Ile Ile Leu Asp Ile Arg Gly 290 295 300Leu Leu Arg Asp Leu Asn Arg
Arg Glu Leu Ile Ser Tyr Lys Asn Lys305 310 315 320Leu Thr Ile Lys
Asp Val Leu Gly Phe Phe Ser Asp Tyr Pro Ile Ile 325 330 335Asp Ile
Lys Lys Asn Leu Val Thr Phe Cys Tyr Lys Glu Gly Val Ile 340 345
350Gln Val Val Ser Gln Lys Ser Ile Gly Asn Lys Lys Ser Lys Gln Leu
355 360 365Leu Glu Lys Leu Ile Glu Asn Lys Pro Ile Ala Leu Val Ser
Ile Asp 370 375 380Leu Gly Gln Thr Asn Pro Val Ser Val Lys Ile Ser
Lys Leu Asn Lys385 390 395 400Ile Asn Asn Lys Ile Ser Ile Glu Ser
Phe Thr Tyr Arg Phe Leu Asn 405 410 415Glu Glu Ile Leu Lys Glu Ile
Glu Lys Tyr Arg Lys Asp Tyr Asp Lys 420 425 430Leu Glu Leu Lys Leu
Ile Asn Glu Ala 435 440117812PRTArtificial SequenceSynthetic
sequence 117Met Asp Met Leu Asp Thr Glu Thr Asn Tyr Ala Thr Glu Thr
Pro Ser1 5 10 15Gln Gln Gln Asp Tyr Ser Pro Lys Pro Pro Lys Lys Asp
Arg Arg Ala 20 25 30Pro Lys Gly Phe Ser Lys Lys Ala Arg Pro Glu Lys
Lys Pro Pro Lys 35 40 45Pro Ile Thr Leu Phe Thr Gln Lys His Phe Ser
Gly Val Arg Phe Leu 50 55 60Lys Arg Val Ile Arg Asp Ala Ser Lys Ile
Leu Lys Leu Ser Glu Ser65 70 75 80Arg Thr Ile Thr Phe Leu Glu Gln
Ala Ile Glu Arg Asp Gly Ser Ala 85 90 95Pro Pro Asp Val Thr Pro Pro
Val His Asn Thr Ile Met Ala Val Thr 100 105 110Arg Pro Phe Glu Glu
Trp Pro Glu Val Ile Leu Ser Lys Ala Leu Gln 115 120 125Lys His Cys
Tyr Ala Leu Thr Lys Lys Ile Lys Ile Lys Thr Trp Pro 130 135 140Lys
Lys Gly Pro Gly Lys Lys Cys Leu Ala Ala Trp Ser Ala Arg Thr145 150
155 160Lys Ile Pro Leu Ile Pro Gly Gln Val Gln Ala Thr Asn Gly Leu
Phe 165 170 175Asp Arg Ile Gly Ser Ile Tyr Asp Gly Val Glu Lys Lys
Val Thr Asn 180 185 190Arg Asn Ala Asn Lys Lys Leu Glu Tyr Asp Glu
Ala Ile Lys Glu Gly 195 200 205Arg Asn Pro Ala Val Pro Glu Tyr Glu
Thr Ala Tyr Asn Ile Asp Gly 210 215 220Thr Leu Ile Asn Lys Pro Gly
Tyr Asn Pro Asn Leu Tyr Ile Thr Gln225 230 235 240Ser Arg Thr Pro
Arg Leu Ile Thr Glu Ala Asp Arg Pro Leu Val Glu 245 250 255Lys Ile
Leu Trp Gln Met Val Glu Lys Lys Thr Gln Ser Arg Asn Gln 260 265
270Ala Arg Arg Ala Arg Leu Glu Lys Ala Ala His Leu Gln Gly Leu Pro
275 280 285Val Pro Lys Phe Val Pro Glu Lys Val Asp Arg Ser Gln Lys
Ile Glu 290 295 300Ile Arg Ile Ile Asp Pro Leu Asp Lys Ile Glu Pro
Tyr Met Pro Gln305 310 315 320Asp Arg Met Ala Ile Lys Ala Ser Gln
Asp Gly His Val Pro Tyr Trp 325 330 335Gln Arg Pro Phe Leu Ser Lys
Arg Arg Asn Arg Arg Val Arg Ala Gly 340 345 350Trp Gly Lys Gln Val
Ser Ser Ile Gln Ala Trp Leu Thr Gly Ala Leu 355 360 365Leu Val Ile
Val Arg Leu Gly Asn Glu Ala Phe Leu Ala Asp Ile Arg 370 375 380Gly
Ala Leu Arg Asn Ala Gln Trp Arg Lys Leu Leu Lys Pro Asp Ala385 390
395 400Thr Tyr Gln Ser Leu Phe Asn Leu Phe Thr Gly Asp Pro Val Val
Asn 405 410 415Thr Arg Thr Asn His Leu Thr Met Ala Tyr Arg Glu Gly
Val Val Asp 420 425 430Ile Val Lys Ser Arg Ser Phe Lys Gly Arg Gln
Thr Arg Glu His Leu 435 440 445Leu Thr Leu Leu Gly Gln Gly Lys Thr
Val Ala Gly Val Ser Phe Asp 450 455 460Leu Gly Gln Lys His Ala Ala
Gly Leu Leu Ala Ala His Phe Gly Leu465 470 475 480Gly Glu Asp Gly
Asn Pro Val Phe Thr Pro Ile Gln Ala Cys Phe Leu 485 490 495Pro Gln
Arg Tyr Leu Asp Ser Leu Thr Asn Tyr Arg Asn Arg Tyr Asp 500 505
510Ala Leu Thr Leu Asp Met Arg Arg Gln Ser Leu Leu Ala Leu Thr Pro
515 520 525Ala Gln Gln Gln Glu Phe Ala Asp Ala Gln Arg Asp Pro Gly
Gly Gln 530 535 540Ala Lys Arg Ala Cys Cys Leu Lys Leu Asn Leu Asn
Pro Asp Glu Ile545 550 555 560Arg Trp Asp Leu Val Ser Gly Ile Ser
Thr Met Ile Ser Asp Leu Tyr 565 570 575Ile Glu Arg Gly Gly Asp Pro
Arg Asp Val His Gln Gln Val Glu Thr 580 585 590Lys Pro Lys Gly Lys
Arg Lys Ser Glu Ile Arg Ile Leu Lys Ile Arg 595 600 605Asp Gly Lys
Trp Ala Tyr Asp Phe Arg Pro Lys Ile Ala Asp Glu Thr 610 615 620Arg
Lys Ala Gln Arg Glu Gln Leu Trp Lys Leu Gln Lys Ala Ser Ser625 630
635 640Glu Phe Glu Arg Leu Ser Arg Tyr Lys Ile Asn Ile Ala Arg Ala
Ile 645 650 655Ala Asn Trp Ala Leu Gln Trp Gly Arg Glu Leu Ser Gly
Cys Asp Ile 660 665 670Val Ile Pro Val Leu Glu Asp Leu Asn Val Gly
Ser Lys Phe Phe Asp 675 680 685Gly Lys Gly Lys Trp Leu Leu Gly Trp
Asp Asn Arg Phe Thr Pro Lys 690 695 700Lys Glu Asn Arg Trp Phe Ile
Lys Val Leu His Lys Ala Val Ala Glu705 710 715 720Leu Ala Pro His
Arg Gly Val Pro Val Tyr Glu Val Met Pro His Arg 725 730 735Thr Ser
Met Thr Cys Pro Ala Cys His Tyr Cys His Pro Thr Asn Arg 740 745
750Glu Gly Asp Arg Phe Glu Cys Gln Ser Cys His Val Val Lys Asn Thr
755 760 765Asp Arg Asp Val Ala Pro Tyr Asn Ile Leu Arg Val Ala Val
Glu Gly 770 775 780Lys Thr Leu Asp Arg Trp Gln Ala Glu Lys Lys Pro
Gln Ala Glu Pro785 790 795 800Asp Arg Pro Met Ile Leu Ile Asp Asn
Gln Glu Ser 805 810118812PRTArtificial SequenceSynthetic sequence
118Met Asp Met Leu Asp Thr Glu Thr Asn Tyr Ala Thr Glu Thr Pro Ser1
5 10 15Gln Gln Gln Asp Tyr Ser Pro Lys Pro Pro Lys Lys Asp Arg Arg
Ala 20 25 30Pro Lys Gly Phe Ser Lys Lys Ala Arg Pro Glu Lys Lys Pro
Pro Lys 35 40 45Pro Ile Thr Leu Phe Thr Gln Lys His Phe Ser Gly Val
Arg Phe Leu 50 55 60Lys Arg Val Ile Arg Asp Ala Ser Lys Ile Leu Lys
Leu Ser Glu Ser65 70 75 80Arg Thr Ile Thr Phe Leu Glu Gln Ala Ile
Glu Arg Asp Gly Ser Ala 85 90
95Pro Pro Asp Val Thr Pro Pro Val His Asn Thr Ile Met Ala Val Thr
100 105 110Arg Pro Phe Glu Glu Trp Pro Glu Val Ile Leu Ser Lys Ala
Leu Gln 115 120 125Lys His Cys Tyr Ala Leu Thr Lys Lys Ile Lys Ile
Lys Thr Trp Pro 130 135 140Lys Lys Gly Pro Gly Lys Lys Cys Leu Ala
Ala Trp Ser Ala Arg Thr145 150 155 160Lys Ile Pro Leu Ile Pro Gly
Gln Val Gln Ala Thr Asn Gly Leu Phe 165 170 175Asp Arg Ile Gly Ser
Ile Tyr Asp Gly Val Glu Lys Lys Val Thr Asn 180 185 190Arg Asn Ala
Asn Lys Lys Leu Glu Tyr Asp Glu Ala Ile Lys Glu Gly 195 200 205Arg
Asn Pro Ala Val Pro Glu Tyr Glu Thr Ala Tyr Asn Ile Asp Gly 210 215
220Thr Leu Ile Asn Lys Pro Gly Tyr Asn Pro Asn Leu Tyr Ile Thr
Gln225 230 235 240Ser Arg Thr Pro Arg Leu Ile Thr Glu Ala Asp Arg
Pro Leu Val Glu 245 250 255Lys Ile Leu Trp Gln Met Val Glu Lys Lys
Thr Gln Ser Arg Asn Gln 260 265 270Ala Arg Arg Ala Arg Leu Glu Lys
Ala Ala His Leu Gln Gly Leu Pro 275 280 285Val Pro Lys Phe Val Pro
Glu Lys Val Asp Arg Ser Gln Lys Ile Glu 290 295 300Ile Arg Ile Ile
Asp Pro Leu Asp Lys Ile Glu Pro Tyr Met Pro Gln305 310 315 320Asp
Arg Met Ala Ile Lys Ala Ser Gln Asp Gly His Val Pro Tyr Trp 325 330
335Gln Arg Pro Phe Leu Ser Lys Arg Arg Asn Arg Arg Val Arg Ala Gly
340 345 350Trp Gly Lys Gln Val Ser Ser Ile Gln Ala Trp Leu Thr Gly
Ala Leu 355 360 365Leu Val Ile Val Arg Leu Gly Asn Glu Ala Phe Leu
Ala Asp Ile Arg 370 375 380Gly Ala Leu Arg Asn Ala Gln Trp Arg Lys
Leu Leu Lys Pro Asp Ala385 390 395 400Thr Tyr Gln Ser Leu Phe Asn
Leu Phe Thr Gly Asp Pro Val Val Asn 405 410 415Thr Arg Thr Asn His
Leu Thr Met Ala Tyr Arg Glu Gly Val Val Asn 420 425 430Ile Val Lys
Ser Arg Ser Phe Lys Gly Arg Gln Thr Arg Glu His Leu 435 440 445Leu
Thr Leu Leu Gly Gln Gly Lys Thr Val Ala Gly Val Ser Phe Asp 450 455
460Leu Gly Gln Lys His Ala Ala Gly Leu Leu Ala Ala His Phe Gly
Leu465 470 475 480Gly Glu Asp Gly Asn Pro Val Phe Thr Pro Ile Gln
Ala Cys Phe Leu 485 490 495Pro Gln Arg Tyr Leu Asp Ser Leu Thr Asn
Tyr Arg Asn Arg Tyr Asp 500 505 510Ala Leu Thr Leu Asp Met Arg Arg
Gln Ser Leu Leu Ala Leu Thr Pro 515 520 525Ala Gln Gln Gln Glu Phe
Ala Asp Ala Gln Arg Asp Pro Gly Gly Gln 530 535 540Ala Lys Arg Ala
Cys Cys Leu Lys Leu Asn Leu Asn Pro Asp Glu Ile545 550 555 560Arg
Trp Asp Leu Val Ser Gly Ile Ser Thr Met Ile Ser Asp Leu Tyr 565 570
575Ile Glu Arg Gly Gly Asp Pro Arg Asp Val His Gln Gln Val Glu Thr
580 585 590Lys Pro Lys Gly Lys Arg Lys Ser Glu Ile Arg Ile Leu Lys
Ile Arg 595 600 605Asp Gly Lys Trp Ala Tyr Asp Phe Arg Pro Lys Ile
Ala Asp Glu Thr 610 615 620Arg Lys Ala Gln Arg Glu Gln Leu Trp Lys
Leu Gln Lys Ala Ser Ser625 630 635 640Glu Phe Glu Arg Leu Ser Arg
Tyr Lys Ile Asn Ile Ala Arg Ala Ile 645 650 655Ala Asn Trp Ala Leu
Gln Trp Gly Arg Glu Leu Ser Gly Cys Asp Ile 660 665 670Val Ile Pro
Val Leu Glu Asp Leu Asn Val Gly Ser Lys Phe Phe Asp 675 680 685Gly
Lys Gly Lys Trp Leu Leu Gly Trp Asp Asn Arg Phe Thr Pro Lys 690 695
700Lys Glu Asn Arg Trp Phe Ile Lys Val Leu His Lys Ala Val Ala
Glu705 710 715 720Leu Ala Pro His Arg Gly Val Pro Val Tyr Glu Val
Met Pro His Arg 725 730 735Thr Ser Met Thr Cys Pro Ala Cys His Tyr
Cys His Pro Thr Asn Arg 740 745 750Glu Gly Asp Arg Phe Glu Cys Gln
Ser Cys His Val Val Lys Asn Thr 755 760 765Asp Arg Asp Val Ala Pro
Tyr Asn Ile Leu Arg Val Ala Val Glu Gly 770 775 780Lys Thr Leu Asp
Arg Trp Gln Ala Glu Lys Lys Pro Gln Ala Glu Pro785 790 795 800Asp
Arg Pro Met Ile Leu Ile Asp Asn Gln Glu Ser 805
810119772PRTArtificial SequenceSynthetic sequence 119Met Ser Asn
Thr Ala Val Ser Thr Arg Glu His Met Ser Asn Lys Thr1 5 10 15Thr Pro
Pro Ser Pro Leu Ser Leu Leu Leu Arg Ala His Phe Pro Gly 20 25 30Leu
Lys Phe Glu Ser Gln Asp Tyr Lys Ile Ala Gly Lys Lys Leu Arg 35 40
45Asp Gly Gly Pro Glu Ala Val Ile Ser Tyr Leu Thr Gly Lys Gly Gln
50 55 60Ala Lys Leu Lys Asp Val Lys Pro Pro Ala Lys Ala Phe Val Ile
Ala65 70 75 80Gln Ser Arg Pro Phe Ile Glu Trp Asp Leu Val Arg Val
Ser Arg Gln 85 90 95Ile Gln Glu Lys Ile Phe Gly Ile Pro Ala Thr Lys
Gly Arg Pro Lys 100 105 110Gln Asp Gly Leu Ser Glu Thr Ala Phe Asn
Glu Ala Val Ala Ser Leu 115 120 125Glu Val Asp Gly Lys Ser Lys Leu
Asn Glu Glu Thr Arg Ala Ala Phe 130 135 140Tyr Glu Val Leu Gly Leu
Asp Ala Pro Ser Leu His Ala Gln Ala Gln145 150 155 160Asn Ala Leu
Ile Lys Ser Ala Ile Ser Ile Arg Glu Gly Val Leu Lys 165 170 175Lys
Val Glu Asn Arg Asn Glu Lys Asn Leu Ser Lys Thr Lys Arg Arg 180 185
190Lys Glu Ala Gly Glu Glu Ala Thr Phe Val Glu Glu Lys Ala His Asp
195 200 205Glu Arg Gly Tyr Leu Ile His Pro Pro Gly Val Asn Gln Thr
Ile Pro 210 215 220Gly Tyr Gln Ala Val Val Ile Lys Ser Cys Pro Ser
Asp Phe Ile Gly225 230 235 240Leu Pro Ser Gly Cys Leu Ala Lys Glu
Ser Ala Glu Ala Leu Thr Asp 245 250 255Tyr Leu Pro His Asp Arg Met
Thr Ile Pro Lys Gly Gln Pro Gly Tyr 260 265 270Val Pro Glu Trp Gln
His Pro Leu Leu Asn Arg Arg Lys Asn Arg Arg 275 280 285Arg Arg Asp
Trp Tyr Ser Ala Ser Leu Asn Lys Pro Lys Ala Thr Cys 290 295 300Ser
Lys Arg Ser Gly Thr Pro Asn Arg Lys Asn Ser Arg Thr Asp Gln305 310
315 320Ile Gln Ser Gly Arg Phe Lys Gly Ala Ile Pro Val Leu Met Arg
Phe 325 330 335Gln Asp Glu Trp Val Ile Ile Asp Ile Arg Gly Leu Leu
Arg Asn Ala 340 345 350Arg Tyr Arg Lys Leu Leu Lys Glu Lys Ser Thr
Ile Pro Asp Leu Leu 355 360 365Ser Leu Phe Thr Gly Asp Pro Ser Ile
Asp Met Arg Gln Gly Val Cys 370 375 380Thr Phe Ile Tyr Lys Ala Gly
Gln Ala Cys Ser Ala Lys Met Val Lys385 390 395 400Thr Lys Asn Ala
Pro Glu Ile Leu Ser Glu Leu Thr Lys Ser Gly Pro 405 410 415Val Val
Leu Val Ser Ile Asp Leu Gly Gln Thr Asn Pro Ile Ala Ala 420 425
430Lys Val Ser Arg Val Thr Gln Leu Ser Asp Gly Gln Leu Ser His Glu
435 440 445Thr Leu Leu Arg Glu Leu Leu Ser Asn Asp Ser Ser Asp Gly
Lys Glu 450 455 460Ile Ala Arg Tyr Arg Val Ala Ser Asp Arg Leu Arg
Asp Lys Leu Ala465 470 475 480Asn Leu Ala Val Glu Arg Leu Ser Pro
Glu His Lys Ser Glu Ile Leu 485 490 495Arg Ala Lys Asn Asp Thr Pro
Ala Leu Cys Lys Ala Arg Val Cys Ala 500 505 510Ala Leu Gly Leu Asn
Pro Glu Met Ile Ala Trp Asp Lys Met Thr Pro 515 520 525Tyr Thr Glu
Phe Leu Ala Thr Ala Tyr Leu Glu Lys Gly Gly Asp Arg 530 535 540Lys
Val Ala Thr Leu Lys Pro Lys Asn Arg Pro Glu Met Leu Arg Arg545 550
555 560Asp Ile Lys Phe Lys Gly Thr Glu Gly Val Arg Ile Glu Val Ser
Pro 565 570 575Glu Ala Ala Glu Ala Tyr Arg Glu Ala Gln Trp Asp Leu
Gln Arg Thr 580 585 590Ser Pro Glu Tyr Leu Arg Leu Ser Thr Trp Lys
Gln Glu Leu Thr Lys 595 600 605Arg Ile Leu Asn Gln Leu Arg His Lys
Ala Ala Lys Ser Ser Gln Cys 610 615 620Glu Val Val Val Met Ala Phe
Glu Asp Leu Asn Ile Lys Met Met His625 630 635 640Gly Asn Gly Lys
Trp Ala Asp Gly Gly Trp Asp Ala Phe Phe Ile Lys 645 650 655Lys Arg
Glu Asn Arg Trp Phe Met Gln Ala Phe His Lys Ser Leu Thr 660 665
670Glu Leu Gly Ala His Lys Gly Val Pro Thr Ile Glu Val Thr Pro His
675 680 685Arg Thr Ser Ile Thr Cys Thr Lys Cys Gly His Cys Asp Lys
Ala Asn 690 695 700Arg Asp Gly Glu Arg Phe Ala Cys Gln Lys Cys Gly
Phe Val Ala His705 710 715 720Ala Asp Leu Glu Ile Ala Thr Asp Asn
Ile Glu Arg Val Ala Leu Thr 725 730 735Gly Lys Pro Met Pro Lys Pro
Glu Ser Glu Arg Ser Gly Asp Ala Lys 740 745 750Lys Ser Val Gly Ala
Arg Lys Ala Ala Phe Lys Pro Glu Glu Asp Ala 755 760 765Glu Ala Ala
Glu 770120717PRTArtificial SequenceSynthetic sequence 120Met Ile
Lys Pro Thr Val Ser Gln Phe Leu Thr Pro Gly Phe Lys Leu1 5 10 15Ile
Arg Asn His Ser Arg Thr Ala Gly Leu Lys Leu Lys Asn Glu Gly 20 25
30Glu Glu Ala Cys Lys Lys Phe Val Arg Glu Asn Glu Ile Pro Lys Asp
35 40 45Glu Cys Pro Asn Phe Gln Gly Gly Pro Ala Ile Ala Asn Ile Ile
Ala 50 55 60Lys Ser Arg Glu Phe Thr Glu Trp Glu Ile Tyr Gln Ser Ser
Leu Ala65 70 75 80Ile Gln Glu Val Ile Phe Thr Leu Pro Lys Asp Lys
Leu Pro Glu Pro 85 90 95Ile Leu Lys Glu Glu Trp Arg Ala Gln Trp Leu
Ser Glu His Gly Leu 100 105 110Asp Thr Val Pro Tyr Lys Glu Ala Ala
Gly Leu Asn Leu Ile Ile Lys 115 120 125Asn Ala Val Asn Thr Tyr Lys
Gly Val Gln Val Lys Val Asp Asn Lys 130 135 140Asn Lys Asn Asn Leu
Ala Lys Ile Asn Arg Lys Asn Glu Ile Ala Lys145 150 155 160Leu Asn
Gly Glu Gln Glu Ile Ser Phe Glu Glu Ile Lys Ala Phe Asp 165 170
175Asp Lys Gly Tyr Leu Leu Gln Lys Pro Ser Pro Asn Lys Ser Ile Tyr
180 185 190Cys Tyr Gln Ser Val Ser Pro Lys Pro Phe Ile Thr Ser Lys
Tyr His 195 200 205Asn Val Asn Leu Pro Glu Glu Tyr Ile Gly Tyr Tyr
Arg Lys Ser Asn 210 215 220Glu Pro Ile Val Ser Pro Tyr Gln Phe Asp
Arg Leu Arg Ile Pro Ile225 230 235 240Gly Glu Pro Gly Tyr Val Pro
Lys Trp Gln Tyr Thr Phe Leu Ser Lys 245 250 255Lys Glu Asn Lys Arg
Arg Lys Leu Ser Lys Arg Ile Lys Asn Val Ser 260 265 270Pro Ile Leu
Gly Ile Ile Cys Ile Lys Lys Asp Trp Cys Val Phe Asp 275 280 285Met
Arg Gly Leu Leu Arg Thr Asn His Trp Lys Lys Tyr His Lys Pro 290 295
300Thr Asp Ser Ile Asn Asp Leu Phe Asp Tyr Phe Thr Gly Asp Pro
Val305 310 315 320Ile Asp Thr Lys Ala Asn Val Val Arg Phe Arg Tyr
Lys Met Glu Asn 325 330 335Gly Ile Val Asn Tyr Lys Pro Val Arg Glu
Lys Lys Gly Lys Glu Leu 340 345 350Leu Glu Asn Ile Cys Asp Gln Asn
Gly Ser Cys Lys Leu Ala Thr Val 355 360 365Asp Val Gly Gln Asn Asn
Pro Val Ala Ile Gly Leu Phe Glu Leu Lys 370 375 380Lys Val Asn Gly
Glu Leu Thr Lys Thr Leu Ile Ser Arg His Pro Thr385 390 395 400Pro
Ile Asp Phe Cys Asn Lys Ile Thr Ala Tyr Arg Glu Arg Tyr Asp 405 410
415Lys Leu Glu Ser Ser Ile Lys Leu Asp Ala Ile Lys Gln Leu Thr Ser
420 425 430Glu Gln Lys Ile Glu Val Asp Asn Tyr Asn Asn Asn Phe Thr
Pro Gln 435 440 445Asn Thr Lys Gln Ile Val Cys Ser Lys Leu Asn Ile
Asn Pro Asn Asp 450 455 460Leu Pro Trp Asp Lys Met Ile Ser Gly Thr
His Phe Ile Ser Glu Lys465 470 475 480Ala Gln Val Ser Asn Lys Ser
Glu Ile Tyr Phe Thr Ser Thr Asp Lys 485 490 495Gly Lys Thr Lys Asp
Val Met Lys Ser Asp Tyr Lys Trp Phe Gln Asp 500 505 510Tyr Lys Pro
Lys Leu Ser Lys Glu Val Arg Asp Ala Leu Ser Asp Ile 515 520 525Glu
Trp Arg Leu Arg Arg Glu Ser Leu Glu Phe Asn Lys Leu Ser Lys 530 535
540Ser Arg Glu Gln Asp Ala Arg Gln Leu Ala Asn Trp Ile Ser Ser
Met545 550 555 560Cys Asp Val Ile Gly Ile Glu Asn Leu Val Lys Lys
Asn Asn Phe Phe 565 570 575Gly Gly Ser Gly Lys Arg Glu Pro Gly Trp
Asp Asn Phe Tyr Lys Pro 580 585 590Lys Lys Glu Asn Arg Trp Trp Ile
Asn Ala Ile His Lys Ala Leu Thr 595 600 605Glu Leu Ser Gln Asn Lys
Gly Lys Arg Val Ile Leu Leu Pro Ala Met 610 615 620Arg Thr Ser Ile
Thr Cys Pro Lys Cys Lys Tyr Cys Asp Ser Lys Asn625 630 635 640Arg
Asn Gly Glu Lys Phe Asn Cys Leu Lys Cys Gly Ile Glu Leu Asn 645 650
655Ala Asp Ile Asp Val Ala Thr Glu Asn Leu Ala Thr Val Ala Ile Thr
660 665 670Ala Gln Ser Met Pro Lys Pro Thr Cys Glu Arg Ser Gly Asp
Ala Lys 675 680 685Lys Pro Val Arg Ala Arg Lys Ala Lys Ala Pro Glu
Phe His Asp Lys 690 695 700Leu Ala Pro Ser Tyr Thr Val Val Leu Arg
Glu Ala Val705 710 715121793PRTArtificial SequenceSynthetic
sequence 121Met Arg Ser Ser Arg Glu Ile Gly Asp Lys Ile Leu Met Arg
Gln Pro1 5 10 15Ala Glu Lys Thr Ala Phe Gln Val Phe Arg Gln Glu Val
Ile Gly Thr 20 25 30Gln Lys Leu Ser Gly Gly Asp Ala Lys Thr Ala Gly
Arg Leu Tyr Lys 35 40 45Gln Gly Lys Met Glu Ala Ala Arg Glu Trp Leu
Leu Lys Gly Ala Arg 50 55 60Asp Asp Val Pro Pro Asn Phe Gln Pro Pro
Ala Lys Cys Leu Val Val65 70 75 80Ala Val Ser His Pro Phe Glu Glu
Trp Asp Ile Ser Lys Thr Asn His 85 90 95Asp Val Gln Ala Tyr Ile Tyr
Ala Gln Pro Leu Gln Ala Glu Gly His 100 105 110Leu Asn Gly Leu Ser
Glu Lys Trp Glu Asp Thr Ser Ala Asp Gln His 115 120 125Lys Leu Trp
Phe Glu Lys Thr Gly Val Pro Asp Arg Gly Leu Pro Val 130 135 140Gln
Ala Ile Asn Lys Ile Ala Lys Ala Ala Val Asn Arg Ala Phe Gly145 150
155 160Val Val Arg Lys Val Glu Asn Arg Asn Glu Lys Arg Arg Ser Arg
Asp 165 170 175Asn Arg Ile Ala Glu His Asn Arg Glu Asn Gly Leu Thr
Glu Val Val 180 185 190Arg Glu Ala Pro Glu Val Ala Thr Asn Ala Asp
Gly Phe Leu Leu His 195 200 205Pro Pro Gly Ile Asp Pro Ser Ile Leu
Ser Tyr Ala Ser Val Ser Pro 210 215 220Val Pro Tyr Asn Ser Ser Lys
His Ser Phe Val Arg Leu Pro Glu Glu225 230 235 240Tyr Gln Ala Tyr
Asn Val Glu Pro Asp Ala Pro Ile Pro Gln Phe Val
245 250 255Val Glu Asp Arg Phe Ala Ile Pro Pro Gly Gln Pro Gly Tyr
Val Pro 260 265 270Glu Trp Gln Arg Leu Lys Cys Ser Thr Asn Lys His
Arg Arg Met Arg 275 280 285Gln Trp Ser Asn Gln Asp Tyr Lys Pro Lys
Ala Gly Arg Arg Ala Lys 290 295 300Pro Leu Glu Phe Gln Ala His Leu
Thr Arg Glu Arg Ala Lys Gly Ala305 310 315 320Leu Leu Val Val Met
Arg Ile Lys Glu Asp Trp Val Val Phe Asp Val 325 330 335Arg Gly Leu
Leu Arg Asn Val Glu Trp Arg Lys Val Leu Ser Glu Glu 340 345 350Ala
Arg Glu Lys Leu Thr Leu Lys Gly Leu Leu Asp Leu Phe Thr Gly 355 360
365Asp Pro Val Ile Asp Thr Lys Arg Gly Ile Val Thr Phe Leu Tyr Lys
370 375 380Ala Glu Ile Thr Lys Ile Leu Ser Lys Arg Thr Val Lys Thr
Lys Asn385 390 395 400Ala Arg Asp Leu Leu Leu Arg Leu Thr Glu Pro
Gly Glu Asp Gly Leu 405 410 415Arg Arg Glu Val Gly Leu Val Ala Val
Asp Leu Gly Gln Thr His Pro 420 425 430Ile Ala Ala Ala Ile Tyr Arg
Ile Gly Arg Thr Ser Ala Gly Ala Leu 435 440 445Glu Ser Thr Val Leu
His Arg Gln Gly Leu Arg Glu Asp Gln Lys Glu 450 455 460Lys Leu Lys
Glu Tyr Arg Lys Arg His Thr Ala Leu Asp Ser Arg Leu465 470 475
480Arg Lys Glu Ala Phe Glu Thr Leu Ser Val Glu Gln Gln Lys Glu Ile
485 490 495Val Thr Val Ser Gly Ser Gly Ala Gln Ile Thr Lys Asp Lys
Val Cys 500 505 510Asn Tyr Leu Gly Val Asp Pro Ser Thr Leu Pro Trp
Glu Lys Met Gly 515 520 525Ser Tyr Thr His Phe Ile Ser Asp Asp Phe
Leu Arg Arg Gly Gly Asp 530 535 540Pro Asn Ile Val His Phe Asp Arg
Gln Pro Lys Lys Gly Lys Val Ser545 550 555 560Lys Lys Ser Gln Arg
Ile Lys Arg Ser Asp Ser Gln Trp Val Gly Arg 565 570 575Met Arg Pro
Arg Leu Ser Gln Glu Thr Ala Lys Ala Arg Met Glu Ala 580 585 590Asp
Trp Ala Ala Gln Asn Glu Asn Glu Glu Tyr Lys Arg Leu Ala Arg 595 600
605Ser Lys Gln Glu Leu Ala Arg Trp Cys Val Asn Thr Leu Leu Gln Asn
610 615 620Thr Arg Cys Ile Thr Gln Cys Asp Glu Ile Val Val Val Ile
Glu Asp625 630 635 640Leu Asn Val Lys Ser Leu His Gly Lys Gly Ala
Arg Glu Pro Gly Trp 645 650 655Asp Asn Phe Phe Thr Pro Lys Thr Glu
Asn Arg Trp Phe Ile Gln Ile 660 665 670Leu His Lys Thr Phe Ser Glu
Leu Pro Lys His Arg Gly Glu His Val 675 680 685Ile Glu Gly Cys Pro
Leu Arg Thr Ser Ile Thr Cys Pro Ala Cys Ser 690 695 700Tyr Cys Asp
Lys Asn Ser Arg Asn Gly Glu Lys Phe Val Cys Val Ala705 710 715
720Cys Gly Ala Thr Phe His Ala Asp Phe Glu Val Ala Thr Tyr Asn Leu
725 730 735Val Arg Leu Ala Thr Thr Gly Met Pro Met Pro Lys Ser Leu
Glu Arg 740 745 750Gln Gly Gly Gly Glu Lys Ala Gly Gly Ala Arg Lys
Ala Arg Lys Lys 755 760 765Ala Lys Gln Val Glu Lys Ile Val Val Gln
Ala Asn Ala Asn Val Thr 770 775 780Met Asn Gly Ala Ser Leu His Ser
Pro785 790122793PRTArtificial SequenceSynthetic sequence 122Met Ser
Ser Leu Pro Thr Pro Leu Glu Leu Leu Lys Gln Lys His Ala1 5 10 15Asp
Leu Phe Lys Gly Leu Gln Phe Ser Ser Lys Asp Asn Lys Met Ala 20 25
30Gly Lys Val Leu Lys Lys Asp Gly Glu Glu Ala Ala Leu Ala Phe Leu
35 40 45Ser Glu Arg Gly Val Ser Arg Gly Glu Leu Pro Asn Phe Arg Pro
Pro 50 55 60Ala Lys Thr Leu Val Val Ala Gln Ser Arg Pro Phe Glu Glu
Phe Pro65 70 75 80Ile Tyr Arg Val Ser Glu Ala Ile Gln Leu Tyr Val
Tyr Ser Leu Ser 85 90 95Val Lys Glu Leu Glu Thr Val Pro Ser Gly Ser
Ser Thr Lys Lys Glu 100 105 110His Gln Arg Phe Phe Gln Asp Ser Ser
Val Pro Asp Phe Gly Tyr Thr 115 120 125Ser Val Gln Gly Leu Asn Lys
Ile Phe Gly Leu Ala Arg Gly Ile Tyr 130 135 140Leu Gly Val Ile Thr
Arg Gly Glu Asn Gln Leu Gln Lys Ala Lys Ser145 150 155 160Lys His
Glu Ala Leu Asn Lys Lys Arg Arg Ala Ser Gly Glu Ala Glu 165 170
175Thr Glu Phe Asp Pro Thr Pro Tyr Glu Tyr Met Thr Pro Glu Arg Lys
180 185 190Leu Ala Lys Pro Pro Gly Val Asn His Ser Ile Met Cys Tyr
Val Asp 195 200 205Ile Ser Val Asp Glu Phe Asp Phe Arg Asn Pro Asp
Gly Ile Val Leu 210 215 220Pro Ser Glu Tyr Ala Gly Tyr Cys Arg Glu
Ile Asn Thr Ala Ile Glu225 230 235 240Lys Gly Thr Val Asp Arg Leu
Gly His Leu Lys Gly Gly Pro Gly Tyr 245 250 255Ile Pro Gly His Gln
Arg Lys Glu Ser Thr Thr Glu Gly Pro Lys Ile 260 265 270Asn Phe Arg
Lys Gly Arg Ile Arg Arg Ser Tyr Thr Ala Leu Tyr Ala 275 280 285Lys
Arg Asp Ser Arg Arg Val Arg Gln Gly Lys Leu Ala Leu Pro Ser 290 295
300Tyr Arg His His Met Met Arg Leu Asn Ser Asn Ala Glu Ser Ala
Ile305 310 315 320Leu Ala Val Ile Phe Phe Gly Lys Asp Trp Val Val
Phe Asp Leu Arg 325 330 335Gly Leu Leu Arg Asn Val Arg Trp Arg Asn
Leu Phe Val Asp Gly Ser 340 345 350Thr Pro Ser Thr Leu Leu Gly Met
Phe Gly Asp Pro Val Ile Asp Pro 355 360 365Lys Arg Gly Val Val Ala
Phe Cys Tyr Lys Glu Gln Ile Val Pro Val 370 375 380Val Ser Lys Ser
Ile Thr Lys Met Val Lys Ala Pro Glu Leu Leu Asn385 390 395 400Lys
Leu Tyr Leu Lys Ser Glu Asp Pro Leu Val Leu Val Ala Ile Asp 405 410
415Leu Gly Gln Thr Asn Pro Val Gly Val Gly Val Tyr Arg Val Met Asn
420 425 430Ala Ser Leu Asp Tyr Glu Val Val Thr Arg Phe Ala Leu Glu
Ser Glu 435 440 445Leu Leu Arg Glu Ile Glu Ser Tyr Arg Gln Arg Thr
Asn Ala Phe Glu 450 455 460Ala Gln Ile Arg Ala Glu Thr Phe Asp Ala
Met Thr Ser Glu Glu Gln465 470 475 480Glu Glu Ile Thr Arg Val Arg
Ala Phe Ser Ala Ser Lys Ala Lys Glu 485 490 495Asn Val Cys His Arg
Phe Gly Met Pro Val Asp Ala Val Asp Trp Ala 500 505 510Thr Met Gly
Ser Asn Thr Ile His Ile Ala Lys Trp Val Met Arg His 515 520 525Gly
Asp Pro Ser Leu Val Glu Val Leu Glu Tyr Arg Lys Asp Asn Glu 530 535
540Ile Lys Leu Asp Lys Asn Gly Val Pro Lys Lys Val Lys Leu Thr
Asp545 550 555 560Lys Arg Ile Ala Asn Leu Thr Ser Ile Arg Leu Arg
Phe Ser Gln Glu 565 570 575Thr Ser Lys His Tyr Asn Asp Thr Met Trp
Glu Leu Arg Arg Lys His 580 585 590Pro Val Tyr Gln Lys Leu Ser Lys
Ser Lys Ala Asp Phe Ser Arg Arg 595 600 605Val Val Asn Ser Ile Ile
Arg Arg Val Asn His Leu Val Pro Arg Ala 610 615 620Arg Ile Val Phe
Ile Ile Glu Asp Leu Lys Asn Leu Gly Lys Val Phe625 630 635 640His
Gly Ser Gly Lys Arg Glu Leu Gly Trp Asp Ser Tyr Phe Glu Pro 645 650
655Lys Ser Glu Asn Arg Trp Phe Ile Gln Val Leu His Lys Ala Phe Ser
660 665 670Glu Thr Gly Lys His Lys Gly Tyr Tyr Ile Ile Glu Cys Trp
Pro Asn 675 680 685Trp Thr Ser Cys Thr Cys Pro Lys Cys Ser Cys Cys
Asp Ser Glu Asn 690 695 700Arg His Gly Glu Val Phe Arg Cys Leu Ala
Cys Gly Tyr Thr Cys Asn705 710 715 720Thr Asp Phe Gly Thr Ala Pro
Asp Asn Leu Val Lys Ile Ala Thr Thr 725 730 735Gly Lys Gly Leu Pro
Gly Pro Lys Lys Arg Cys Lys Gly Ser Ser Lys 740 745 750Gly Lys Asn
Pro Lys Ile Ala Arg Ser Ser Glu Thr Gly Val Ser Val 755 760 765Thr
Glu Ser Gly Ala Pro Lys Val Lys Lys Ser Ser Pro Thr Gln Thr 770 775
780Ser Gln Ser Ser Ser Gln Ser Ala Pro785 790123717PRTArtificial
SequenceSynthetic sequence 123Met Ile Lys Pro Thr Val Ser Gln Phe
Leu Thr Pro Gly Phe Lys Leu1 5 10 15Ile Arg Asn His Ser Arg Thr Ala
Gly Leu Lys Leu Lys Asn Glu Gly 20 25 30Glu Glu Ala Cys Lys Lys Phe
Val Arg Glu Asn Glu Ile Pro Lys Asp 35 40 45Glu Cys Pro Asn Phe Gln
Gly Gly Pro Ala Ile Ala Asn Ile Ile Ala 50 55 60Lys Ser Arg Glu Phe
Thr Glu Trp Glu Ile Tyr Gln Ser Ser Leu Ala65 70 75 80Ile Gln Glu
Val Ile Phe Thr Leu Pro Lys Asp Lys Leu Pro Glu Pro 85 90 95Ile Leu
Lys Glu Glu Trp Arg Ala Gln Trp Leu Ser Glu His Gly Leu 100 105
110Asp Thr Val Pro Tyr Lys Glu Ala Ala Gly Leu Asn Leu Ile Ile Lys
115 120 125Asn Ala Val Asn Thr Tyr Lys Gly Val Gln Val Lys Val Asp
Asn Lys 130 135 140Asn Lys Asn Asn Leu Ala Lys Ile Asn Arg Lys Asn
Glu Ile Ala Lys145 150 155 160Leu Asn Gly Glu Gln Glu Ile Ser Phe
Glu Glu Ile Lys Ala Phe Asp 165 170 175Asp Lys Gly Tyr Leu Leu Gln
Lys Pro Ser Pro Asn Lys Ser Ile Tyr 180 185 190Cys Tyr Gln Ser Val
Ser Pro Lys Pro Phe Ile Thr Ser Lys Tyr His 195 200 205Asn Val Asn
Leu Pro Glu Glu Tyr Ile Gly Tyr Tyr Arg Lys Ser Asn 210 215 220Glu
Pro Ile Val Ser Pro Tyr Gln Phe Asp Arg Leu Arg Ile Pro Ile225 230
235 240Gly Glu Pro Gly Tyr Val Pro Lys Trp Gln Tyr Thr Phe Leu Ser
Lys 245 250 255Lys Glu Asn Lys Arg Arg Lys Leu Ser Lys Arg Ile Lys
Asn Val Ser 260 265 270Pro Ile Leu Gly Ile Ile Cys Ile Lys Lys Asp
Trp Cys Val Phe Asp 275 280 285Met Arg Gly Leu Leu Arg Thr Asn His
Trp Lys Lys Tyr His Lys Pro 290 295 300Thr Asp Ser Ile Asn Asp Leu
Phe Asp Tyr Phe Thr Gly Asp Pro Val305 310 315 320Ile Asp Thr Lys
Ala Asn Val Val Arg Phe Arg Tyr Lys Met Glu Asn 325 330 335Gly Ile
Val Asn Tyr Lys Pro Val Arg Glu Lys Lys Gly Lys Glu Leu 340 345
350Leu Glu Asn Ile Cys Asp Gln Asn Gly Ser Cys Lys Leu Ala Thr Val
355 360 365Asp Val Gly Gln Asn Asn Pro Val Ala Ile Gly Leu Phe Glu
Leu Lys 370 375 380Lys Val Asn Gly Glu Leu Thr Lys Thr Leu Ile Ser
Arg His Pro Thr385 390 395 400Pro Ile Asp Phe Cys Asn Lys Ile Thr
Ala Tyr Arg Glu Arg Tyr Asp 405 410 415Lys Leu Glu Ser Ser Ile Lys
Leu Asp Ala Ile Lys Gln Leu Thr Ser 420 425 430Glu Gln Lys Ile Glu
Val Asp Asn Tyr Asn Asn Asn Phe Thr Pro Gln 435 440 445Asn Thr Lys
Gln Ile Val Cys Ser Lys Leu Asn Ile Asn Pro Asn Asp 450 455 460Leu
Pro Trp Asp Lys Met Ile Ser Gly Thr His Phe Ile Ser Glu Lys465 470
475 480Ala Gln Val Ser Asn Lys Ser Glu Ile Tyr Phe Thr Ser Thr Asp
Lys 485 490 495Gly Lys Thr Lys Asp Val Met Lys Ser Asp Tyr Lys Trp
Phe Gln Asp 500 505 510Tyr Lys Pro Lys Leu Ser Lys Glu Val Arg Asp
Ala Leu Ser Asp Ile 515 520 525Glu Trp Arg Leu Arg Arg Glu Ser Leu
Glu Phe Asn Lys Leu Ser Lys 530 535 540Ser Arg Glu Gln Asp Ala Arg
Gln Leu Ala Asn Trp Ile Ser Ser Met545 550 555 560Cys Asp Val Ile
Gly Ile Glu Asn Leu Val Lys Lys Asn Asn Phe Phe 565 570 575Gly Gly
Ser Gly Lys Arg Glu Pro Gly Trp Asp Asn Phe Tyr Lys Pro 580 585
590Lys Lys Glu Asn Arg Trp Trp Ile Asn Ala Ile His Lys Ala Leu Thr
595 600 605Glu Leu Ser Gln Asn Lys Gly Lys Arg Val Ile Leu Leu Pro
Ala Met 610 615 620Arg Thr Ser Ile Thr Cys Pro Lys Cys Lys Tyr Cys
Asp Ser Lys Asn625 630 635 640Arg Asn Gly Glu Lys Phe Asn Cys Leu
Lys Cys Gly Ile Glu Leu Asn 645 650 655Ala Asp Ile Asp Val Ala Thr
Glu Asn Leu Ala Thr Val Ala Ile Thr 660 665 670Ala Gln Ser Met Pro
Lys Pro Thr Cys Glu Arg Ser Gly Asp Ala Lys 675 680 685Lys Pro Val
Arg Ala Arg Lys Ala Lys Ala Pro Glu Phe His Asp Lys 690 695 700Leu
Ala Pro Ser Tyr Thr Val Val Leu Arg Glu Ala Val705 710
715124772PRTArtificial SequenceSynthetic sequence 124Met Ser Asn
Thr Ala Val Ser Thr Arg Glu His Met Ser Asn Lys Thr1 5 10 15Thr Pro
Pro Ser Pro Leu Ser Leu Leu Leu Arg Ala His Phe Pro Gly 20 25 30Leu
Lys Phe Glu Ser Gln Asp Tyr Lys Ile Ala Gly Lys Lys Leu Arg 35 40
45Asp Gly Gly Pro Glu Ala Val Ile Ser Tyr Leu Thr Gly Lys Gly Gln
50 55 60Ala Lys Leu Lys Asp Val Lys Pro Pro Ala Lys Ala Phe Val Ile
Ala65 70 75 80Gln Ser Arg Pro Phe Ile Glu Trp Asp Leu Val Arg Val
Ser Arg Gln 85 90 95Ile Gln Glu Lys Ile Phe Gly Ile Pro Ala Thr Lys
Gly Arg Pro Lys 100 105 110Gln Asp Gly Leu Ser Glu Thr Ala Phe Asn
Glu Ala Val Ala Ser Leu 115 120 125Glu Val Asp Gly Lys Ser Lys Leu
Asn Glu Glu Thr Arg Ala Ala Phe 130 135 140Tyr Glu Val Leu Gly Leu
Asp Ala Pro Ser Leu His Ala Gln Ala Gln145 150 155 160Asn Ala Leu
Ile Lys Ser Ala Ile Ser Ile Arg Glu Gly Val Leu Lys 165 170 175Lys
Val Glu Asn Arg Asn Glu Lys Asn Leu Ser Lys Thr Lys Arg Arg 180 185
190Lys Glu Ala Gly Glu Glu Ala Thr Phe Val Glu Glu Lys Ala His Asp
195 200 205Glu Arg Gly Tyr Leu Ile His Pro Pro Gly Val Asn Gln Thr
Ile Pro 210 215 220Gly Tyr Gln Ala Val Val Ile Lys Ser Cys Pro Ser
Asp Phe Ile Gly225 230 235 240Leu Pro Ser Gly Cys Leu Ala Lys Glu
Ser Ala Glu Ala Leu Thr Asp 245 250 255Tyr Leu Pro His Asp Arg Met
Thr Ile Pro Lys Gly Gln Pro Gly Tyr 260 265 270Val Pro Glu Trp Gln
His Pro Leu Leu Asn Arg Arg Lys Asn Arg Arg 275 280 285Arg Arg Asp
Trp Tyr Ser Ala Ser Leu Asn Lys Pro Lys Ala Thr Cys 290 295 300Ser
Lys Arg Ser Gly Thr Pro Asn Arg Lys Asn Ser Arg Thr Asp Gln305 310
315 320Ile Gln Ser Gly Arg Phe Lys Gly Ala Ile Pro Val Leu Met Arg
Phe 325 330 335Gln Asp Glu Trp Val Ile Ile Asp Ile Arg Gly Leu Leu
Arg Asn Ala 340 345 350Arg Tyr Arg Lys Leu Leu Lys Glu Lys Ser Thr
Ile Pro Asp Leu Leu 355 360 365Ser Leu Phe Thr Gly Asp Pro Ser Ile
Asp Met Arg Gln Gly Val Cys 370 375 380Thr Phe Ile Tyr Lys Ala Gly
Gln Ala Cys Ser Ala Lys Met Val Lys385 390 395 400Thr Lys Asn Ala
Pro Glu Ile Leu Ser Glu
Leu Thr Lys Ser Gly Pro 405 410 415Val Val Leu Val Ser Ile Asp Leu
Gly Gln Thr Asn Pro Ile Ala Ala 420 425 430Lys Val Ser Arg Val Thr
Gln Leu Ser Asp Gly Gln Leu Ser His Glu 435 440 445Thr Leu Leu Arg
Glu Leu Leu Ser Asn Asp Ser Ser Asp Gly Lys Glu 450 455 460Ile Ala
Arg Tyr Arg Val Ala Ser Asp Arg Leu Arg Asp Lys Leu Ala465 470 475
480Asn Leu Ala Val Glu Arg Leu Ser Pro Glu His Lys Ser Glu Ile Leu
485 490 495Arg Ala Lys Asn Asp Thr Pro Ala Leu Cys Lys Ala Arg Val
Cys Ala 500 505 510Ala Leu Gly Leu Asn Pro Glu Met Ile Ala Trp Asp
Lys Met Thr Pro 515 520 525Tyr Thr Glu Phe Leu Ala Thr Ala Tyr Leu
Glu Lys Gly Gly Asp Arg 530 535 540Lys Val Ala Thr Leu Lys Pro Lys
Asn Arg Pro Glu Met Leu Arg Arg545 550 555 560Asp Ile Lys Phe Lys
Gly Thr Glu Gly Val Arg Ile Glu Val Ser Pro 565 570 575Glu Ala Ala
Glu Ala Tyr Arg Glu Ala Gln Trp Asp Leu Gln Arg Thr 580 585 590Ser
Pro Glu Tyr Leu Arg Leu Ser Thr Trp Lys Gln Glu Leu Thr Lys 595 600
605Arg Ile Leu Asn Gln Leu Arg His Lys Ala Ala Lys Ser Ser Gln Cys
610 615 620Glu Val Val Val Met Ala Phe Glu Asp Leu Asn Ile Lys Met
Met His625 630 635 640Gly Asn Gly Lys Trp Ala Asp Gly Gly Trp Asp
Ala Phe Phe Ile Lys 645 650 655Lys Arg Glu Asn Arg Trp Phe Met Gln
Ala Phe His Lys Ser Leu Thr 660 665 670Glu Leu Gly Ala His Lys Gly
Val Pro Thr Ile Glu Val Thr Pro His 675 680 685Arg Thr Ser Ile Thr
Cys Thr Lys Cys Gly His Cys Asp Lys Ala Asn 690 695 700Arg Asp Gly
Glu Arg Phe Ala Cys Gln Lys Cys Gly Phe Val Ala His705 710 715
720Ala Asp Leu Glu Ile Ala Thr Asp Asn Ile Glu Arg Val Ala Leu Thr
725 730 735Gly Lys Pro Met Pro Lys Pro Glu Ser Glu Arg Ser Gly Asp
Ala Lys 740 745 750Lys Ser Val Gly Ala Arg Lys Ala Ala Phe Lys Pro
Glu Glu Asp Ala 755 760 765Glu Ala Ala Glu 770125765PRTArtificial
SequenceSynthetic sequence 125Met Tyr Ser Leu Glu Met Ala Asp Leu
Lys Ser Glu Pro Ser Leu Leu1 5 10 15Ala Lys Leu Leu Arg Asp Arg Phe
Pro Gly Lys Tyr Trp Leu Pro Lys 20 25 30Tyr Trp Lys Leu Ala Glu Lys
Lys Arg Leu Thr Gly Gly Glu Glu Ala 35 40 45Ala Cys Glu Tyr Met Ala
Asp Lys Gln Leu Asp Ser Pro Pro Pro Asn 50 55 60Phe Arg Pro Pro Ala
Arg Cys Val Ile Leu Ala Lys Ser Arg Pro Phe65 70 75 80Glu Asp Trp
Pro Val His Arg Val Ala Ser Lys Ala Gln Ser Phe Val 85 90 95Ile Gly
Leu Ser Glu Gln Gly Phe Ala Ala Leu Arg Ala Ala Pro Pro 100 105
110Ser Thr Ala Asp Ala Arg Arg Asp Trp Leu Arg Ser His Gly Ala Ser
115 120 125Glu Asp Asp Leu Met Ala Leu Glu Ala Gln Leu Leu Glu Thr
Ile Met 130 135 140Gly Asn Ala Ile Ser Leu His Gly Gly Val Leu Lys
Lys Ile Asp Asn145 150 155 160Ala Asn Val Lys Ala Ala Lys Arg Leu
Ser Gly Arg Asn Glu Ala Arg 165 170 175Leu Asn Lys Gly Leu Gln Glu
Leu Pro Pro Glu Gln Glu Gly Ser Ala 180 185 190Tyr Gly Ala Asp Gly
Leu Leu Val Asn Pro Pro Gly Leu Asn Leu Asn 195 200 205Ile Tyr Cys
Arg Lys Ser Cys Cys Pro Lys Pro Val Lys Asn Thr Ala 210 215 220Arg
Phe Val Gly His Tyr Pro Gly Tyr Leu Arg Asp Ser Asp Ser Ile225 230
235 240Leu Ile Ser Gly Thr Met Asp Arg Leu Thr Ile Ile Glu Gly Met
Pro 245 250 255Gly His Ile Pro Ala Trp Gln Arg Glu Gln Gly Leu Val
Lys Pro Gly 260 265 270Gly Arg Arg Arg Arg Leu Ser Gly Ser Glu Ser
Asn Met Arg Gln Lys 275 280 285Val Asp Pro Ser Thr Gly Pro Arg Arg
Ser Thr Arg Ser Gly Thr Val 290 295 300Asn Arg Ser Asn Gln Arg Thr
Gly Arg Asn Gly Asp Pro Leu Leu Val305 310 315 320Glu Ile Arg Met
Lys Glu Asp Trp Val Leu Leu Asp Ala Arg Gly Leu 325 330 335Leu Arg
Asn Leu Arg Trp Arg Glu Ser Lys Arg Gly Leu Ser Cys Asp 340 345
350His Glu Asp Leu Ser Leu Ser Gly Leu Leu Ala Leu Phe Ser Gly Asp
355 360 365Pro Val Ile Asp Pro Val Arg Asn Glu Val Val Phe Leu Tyr
Gly Glu 370 375 380Gly Ile Ile Pro Val Arg Ser Thr Lys Pro Val Gly
Thr Arg Gln Ser385 390 395 400Lys Lys Leu Leu Glu Arg Gln Ala Ser
Met Gly Pro Leu Thr Leu Ile 405 410 415Ser Cys Asp Leu Gly Gln Thr
Asn Leu Ile Ala Gly Arg Ala Ser Ala 420 425 430Ile Ser Leu Thr His
Gly Ser Leu Gly Val Arg Ser Ser Val Arg Ile 435 440 445Glu Leu Asp
Pro Glu Ile Ile Lys Ser Phe Glu Arg Leu Arg Lys Asp 450 455 460Ala
Asp Arg Leu Glu Thr Glu Ile Leu Thr Ala Ala Lys Glu Thr Leu465 470
475 480Ser Asp Glu Gln Arg Gly Glu Val Asn Ser His Glu Lys Asp Ser
Pro 485 490 495Gln Thr Ala Lys Ala Ser Leu Cys Arg Glu Leu Gly Leu
His Pro Pro 500 505 510Ser Leu Pro Trp Gly Gln Met Gly Pro Ser Thr
Thr Phe Ile Ala Asp 515 520 525Met Leu Ile Ser His Gly Arg Asp Asp
Asp Ala Phe Leu Ser His Gly 530 535 540Glu Phe Pro Thr Leu Glu Lys
Arg Lys Lys Phe Asp Lys Arg Phe Cys545 550 555 560Leu Glu Ser Arg
Pro Leu Leu Ser Ser Glu Thr Arg Lys Ala Leu Asn 565 570 575Glu Ser
Leu Trp Glu Val Lys Arg Thr Ser Ser Glu Tyr Ala Arg Leu 580 585
590Ser Gln Arg Lys Lys Glu Met Ala Arg Arg Ala Val Asn Phe Val Val
595 600 605Glu Ile Ser Arg Arg Lys Thr Gly Leu Ser Asn Val Ile Val
Asn Ile 610 615 620Glu Asp Leu Asn Val Arg Ile Phe His Gly Gly Gly
Lys Gln Ala Pro625 630 635 640Gly Trp Asp Gly Phe Phe Arg Pro Lys
Ser Glu Asn Arg Trp Phe Ile 645 650 655Gln Ala Ile His Lys Ala Phe
Ser Asp Leu Ala Ala His His Gly Ile 660 665 670Pro Val Ile Glu Ser
Asp Pro Gln Arg Thr Ser Met Thr Cys Pro Glu 675 680 685Cys Gly His
Cys Asp Ser Lys Asn Arg Asn Gly Val Arg Phe Leu Cys 690 695 700Lys
Gly Cys Gly Ala Ser Met Asp Ala Asp Phe Asp Ala Ala Cys Arg705 710
715 720Asn Leu Glu Arg Val Ala Leu Thr Gly Lys Pro Met Pro Lys Pro
Ser 725 730 735Thr Ser Cys Glu Arg Leu Leu Ser Ala Thr Thr Gly Lys
Val Cys Ser 740 745 750Asp His Ser Leu Ser His Asp Ala Ile Glu Lys
Ala Ser 755 760 765126766PRTArtificial SequenceSynthetic sequence
126Met Glu Lys Glu Ile Thr Glu Leu Thr Lys Ile Arg Arg Glu Phe Pro1
5 10 15Asn Lys Lys Phe Ser Ser Thr Asp Met Lys Lys Ala Gly Lys Leu
Leu 20 25 30Lys Ala Glu Gly Pro Asp Ala Val Arg Asp Phe Leu Asn Ser
Cys Gln 35 40 45Glu Ile Ile Gly Asp Phe Lys Pro Pro Val Lys Thr Asn
Ile Val Ser 50 55 60Ile Ser Arg Pro Phe Glu Glu Trp Pro Val Ser Met
Val Gly Arg Ala65 70 75 80Ile Gln Glu Tyr Tyr Phe Ser Leu Thr Lys
Glu Glu Leu Glu Ser Val 85 90 95His Pro Gly Thr Ser Ser Glu Asp His
Lys Ser Phe Phe Asn Ile Thr 100 105 110Gly Leu Ser Asn Tyr Asn Tyr
Thr Ser Val Gln Gly Leu Asn Leu Ile 115 120 125Phe Lys Asn Ala Lys
Ala Ile Tyr Asp Gly Thr Leu Val Lys Ala Asn 130 135 140Asn Lys Asn
Lys Lys Leu Glu Lys Lys Phe Asn Glu Ile Asn His Lys145 150 155
160Arg Ser Leu Glu Gly Leu Pro Ile Ile Thr Pro Asp Phe Glu Glu Pro
165 170 175Phe Asp Glu Asn Gly His Leu Asn Asn Pro Pro Gly Ile Asn
Arg Asn 180 185 190Ile Tyr Gly Tyr Gln Gly Cys Ala Ala Lys Val Phe
Val Pro Ser Lys 195 200 205His Lys Met Val Ser Leu Pro Lys Glu Tyr
Glu Gly Tyr Asn Arg Asp 210 215 220Pro Asn Leu Ser Leu Ala Gly Phe
Arg Asn Arg Leu Glu Ile Pro Glu225 230 235 240Gly Glu Pro Gly His
Val Pro Trp Phe Gln Arg Met Asp Ile Pro Glu 245 250 255Gly Gln Ile
Gly His Val Asn Lys Ile Gln Arg Phe Asn Phe Val His 260 265 270Gly
Lys Asn Ser Gly Lys Val Lys Phe Ser Asp Lys Thr Gly Arg Val 275 280
285Lys Arg Tyr His His Ser Lys Tyr Lys Asp Ala Thr Lys Pro Tyr Lys
290 295 300Phe Leu Glu Glu Ser Lys Lys Val Ser Ala Leu Asp Ser Ile
Leu Ala305 310 315 320Ile Ile Thr Ile Gly Asp Asp Trp Val Val Phe
Asp Ile Arg Gly Leu 325 330 335Tyr Arg Asn Val Phe Tyr Arg Glu Leu
Ala Gln Lys Gly Leu Thr Ala 340 345 350Val Gln Leu Leu Asp Leu Phe
Thr Gly Asp Pro Val Ile Asp Pro Lys 355 360 365Lys Gly Val Val Thr
Phe Ser Tyr Lys Glu Gly Val Val Pro Val Phe 370 375 380Ser Gln Lys
Ile Val Pro Arg Phe Lys Ser Arg Asp Thr Leu Glu Lys385 390 395
400Leu Thr Ser Gln Gly Pro Val Ala Leu Leu Ser Val Asp Leu Gly Gln
405 410 415Asn Glu Pro Val Ala Ala Arg Val Cys Ser Leu Lys Asn Ile
Asn Asp 420 425 430Lys Ile Thr Leu Asp Asn Ser Cys Arg Ile Ser Phe
Leu Asp Asp Tyr 435 440 445Lys Lys Gln Ile Lys Asp Tyr Arg Asp Ser
Leu Asp Glu Leu Glu Ile 450 455 460Lys Ile Arg Leu Glu Ala Ile Asn
Ser Leu Glu Thr Asn Gln Gln Val465 470 475 480Glu Ile Arg Asp Leu
Asp Val Phe Ser Ala Asp Arg Ala Lys Ala Asn 485 490 495Thr Val Asp
Met Phe Asp Ile Asp Pro Asn Leu Ile Ser Trp Asp Ser 500 505 510Met
Ser Asp Ala Arg Val Ser Thr Gln Ile Ser Asp Leu Tyr Leu Lys 515 520
525Asn Gly Gly Asp Glu Ser Arg Val Tyr Phe Glu Ile Asn Asn Lys Arg
530 535 540Ile Lys Arg Ser Asp Tyr Asn Ile Ser Gln Leu Val Arg Pro
Lys Leu545 550 555 560Ser Asp Ser Thr Arg Lys Asn Leu Asn Asp Ser
Ile Trp Lys Leu Lys 565 570 575Arg Thr Ser Glu Glu Tyr Leu Lys Leu
Ser Lys Arg Lys Leu Glu Leu 580 585 590Ser Arg Ala Val Val Asn Tyr
Thr Ile Arg Gln Ser Lys Leu Leu Ser 595 600 605Gly Ile Asn Asp Ile
Val Ile Ile Leu Glu Asp Leu Asp Val Lys Lys 610 615 620Lys Phe Asn
Gly Arg Gly Ile Arg Asp Ile Gly Trp Asp Asn Phe Phe625 630 635
640Ser Ser Arg Lys Glu Asn Arg Trp Phe Ile Pro Ala Phe His Lys Thr
645 650 655Phe Ser Glu Leu Ser Ser Asn Arg Gly Leu Cys Val Ile Glu
Val Asn 660 665 670Pro Ala Trp Thr Ser Ala Thr Cys Pro Asp Cys Gly
Phe Cys Ser Lys 675 680 685Glu Asn Arg Asp Gly Ile Asn Phe Thr Cys
Arg Lys Cys Gly Val Ser 690 695 700Tyr His Ala Asp Ile Asp Val Ala
Thr Leu Asn Ile Ala Arg Val Ala705 710 715 720Val Leu Gly Lys Pro
Met Ser Gly Pro Ala Asp Arg Glu Arg Leu Gly 725 730 735Asp Thr Lys
Lys Pro Arg Val Ala Arg Ser Arg Lys Thr Met Lys Arg 740 745 750Lys
Asp Ile Ser Asn Ser Thr Val Glu Ala Met Val Thr Ala 755 760
76512736DNAArtificial SequenceSynthetic sequence 127gtctcgacta
atcgagcaat cgtttgagat ctctcc 3612836DNAArtificial SequenceSynthetic
sequence 128ggagagatct caaacgattg ctcgattagt cgagac
3612936DNAArtificial SequenceSynthetic sequence 129gtcggaacgc
tcaacgattg cccctcacga ggggac 3613036DNAArtificial SequenceSynthetic
sequence 130gtcccctcgt gaggggcaat cgttgagcgt tccgac
3613136DNAArtificial SequenceSynthetic sequence 131gtcccagcgt
actgggcaat caatagtcgt tttggt 3613236DNAArtificial SequenceSynthetic
sequence 132accaaaacga ctattgattg cccagtacgc tgggac
3613337DNAArtificial SequenceSynthetic sequence 133ggatccaatc
ctttttgatt gcccaattcg ttgggac 3713436DNAArtificial
SequenceSynthetic sequence 134ggatctgagg atcattattg ctcgttacga
cgagac 3613536DNAArtificial SequenceSynthetic sequence
135gtctcgtcgt aacgagcaat aatgatcctc agatcc 3613636DNAArtificial
SequenceSynthetic sequence 136gtctcagcgt actgagcaat caaaaggttt
cgcagg 3613736DNAArtificial SequenceSynthetic sequence
137cctgcgaaac cttttgattg ctcagtacgc tgagac 3613836DNAArtificial
SequenceSynthetic sequence 138gtctcctcgt aaggagcaat ctattagtct
tgaaag 3613936DNAArtificial SequenceSynthetic sequence
139ctttcaagac taatagattg ctccttacga ggagac 3614036DNAArtificial
SequenceSynthetic sequence 140gtctcggcgc accgagcaat cagcgaggtc
ttctac 3614136DNAArtificial SequenceSynthetic sequence
141gtagaagacc tcgctgattg ctcggtgcgc cgagac 3614236DNAArtificial
SequenceSynthetic sequence 142gtctcctcgt aaggagcaat ctattagtct
tgaaag 3614336DNAArtificial SequenceSynthetic sequence
143ctttcaagac taatagattg ctccttacga ggagac 3614436DNAArtificial
SequenceSynthetic sequence 144gtctcagcgt actgagcaat caaaaggttt
cgcagg 3614536DNAArtificial SequenceSynthetic sequence
145cctgcgaaac cttttgattg ctcagtacgc tgagac 3614636DNAArtificial
SequenceSynthetic sequence 146accaaaacga ctattgattg cccagtacgc
tgggac 3614737DNAArtificial SequenceSynthetic sequence
147gtcccaacga attgggcaat caaaaaggat tggatcc 3714837DNAArtificial
SequenceSynthetic sequence 148ggatccaatc ctttttgatt gcccaattcg
ttgggac 3714936DNAArtificial SequenceSynthetic sequence
149gtctcagcgt actgagcaat caaaaggttt cgcagg 3615036DNAArtificial
SequenceSynthetic sequence 150cctgcgaaac cttttgattg ctcagtacgc
tgagac 3615136DNAArtificial SequenceSynthetic sequence
151gtctcgacta atcgagcaat cgtttgagat ctctcc 3615236DNAArtificial
SequenceSynthetic sequence 152ggagagatct caaacgattg ctcgattagt
cgagac 3615336DNAArtificial SequenceSynthetic sequence
153gtcggaacgc tcaacgattg cccctcacga ggggac 3615436DNAArtificial
SequenceSynthetic sequence 154gtcccctcgt gaggggcaat cgttgagcgt
tccgac 3615536DNAArtificial SequenceSynthetic sequence
155gtcgcggcgt accgcgcaat gagagtctgt tgccat 3615636DNAArtificial
SequenceSynthetic sequence 156atggcaacag actctcattg cgcggtacgc
cgcgac 3615736DNAArtificial SequenceSynthetic sequence
157gtctcctcgt aaggagcaat ctattagtct tgaaag 3615836DNAArtificial
SequenceSynthetic sequence 158ctttcaagac taatagattg ctccttacga
ggagac 3615936DNAArtificial SequenceSynthetic sequence
159gtctcggcgc accgagcaat cagcgaggtc ttctac 3616036DNAArtificial
SequenceSynthetic sequence 160gtagaagacc tcgctgattg ctcggtgcgc
cgagac
361617180DNAArtificial SequenceSynthetic sequence 161atgccaaagc
cagccgtgga gtctgagttt tctaaggtac tcaagaagca ctttccgggc 60gagcgattta
ggtctagcta catgaagcgg ggtggtaaaa tcttggcagc ccagggtgaa
120gaagcggtcg tcgcgtatct gcaaggcaag tccgaggagg aacccccgaa
ttttcagccg 180ccggcgaaat gtcatgttgt tacgaaatca cgagatttcg
ccgagtggcc aattatgaag 240gcctccgaag caatccaaag gtatatctat
gcgctctcta cgacggaacg ggcagcttgc 300aagcctggca aatcttcaga
gtcccacgcg gcctggttcg cggcaactgg cgtgtcaaac 360cacggttata
gccatgttca aggcctcaat cttatcttcg accacacgct gggaagatac
420gatggtgttc tgaaaaaggt gcagctgaga aatgagaaag cccgcgcccg
gctggaaagt 480atcaacgcct ctcgagccga cgaaggactt ccagaaataa
aggcagagga ggaagaggtc 540gctacaaatg aaaccggaca ccttttgcag
cctccgggga tcaacccaag tttctacgtt 600taccagacta tttctccgca
ggcttacagg ccgcgagatg agattgtact gccgcccgag 660tatgccggct
acgtccgaga tccgaacgcc cctatccccc ttggcgtggt tcggaatcgg
720tgcgatattc agaagggatg ccctggatac atccccgaat ggcaaagaga
ggcaggtact 780gcaatttccc ctaagacggg taaagccgtc accgttcccg
gcctcagtcc aaaaaaaaat 840aaacgaatgc gacgatactg gaggtccgag
aaagagaagg cccaagatgc actgctcgtt 900actgtgagaa tcggcactga
ctgggtcgta atcgacgttc gaggtttgct gcggaatgcg 960cggtggcgca
ccattgcgcc caaggatata tccttgaatg ccctcttgga tctctttaca
1020ggcgacccgg tcatagatgt tcggagaaac attgtgactt tcacctacac
tctggacgct 1080tgcggtacat atgctcgcaa atggactctc aaagggaaac
agactaaggc aaccctcgat 1140aagttgaccg caacccagac cgtggccctg
gtagcaatag accttggaca aaccaatccc 1200ataagtgcgg gtatcagtag
ggtcacgcaa gaaaacgggg cacttcaatg tgaacctctg 1260gatcggttca
ctctccctga tgatctgctc aaggatatct ccgcgtaccg aatcgcttgg
1320gatcgcaacg aggaggaact gagggctagg tccgtcgaag cgctcccaga
agctcaacaa 1380gctgaagtga gggctctgga cggcgtttct aaagaaaccg
ccaggaccca gctctgcgcg 1440gacttcggcc ttgatcccaa acggctgcct
tgggataaaa tgagcagcaa caccactttc 1500atcagtgaag cgttgcttag
taattctgtg tctagagatc aggttttttt tactcctgcg 1560cctaaaaagg
gagcaaagaa aaaagccccc gttgaagtta tgcggaagga taggacctgg
1620gcgagggcct ataaaccacg gctcagtgtg gaagcccaaa agctgaaaaa
tgaggccttg 1680tgggctctca agcgcacttc tccagaatac ctcaagctga
gtcggagaaa agaggagctt 1740tgtaggcgaa gtattaacta cgtcattgaa
aaaacaagac ggaggacaca atgtcagatc 1800gtgatacctg tcatagagga
cttgaatgtg cgattctttc acggttcagg gaagcgcctg 1860cctggctggg
ataatttttt cactgcgaag aaggagaaca ggtggtttat acagggcctc
1920cacaaagcat tcagcgactt gcgaactcat cgctccttct acgtattcga
agtccgcccg 1980gagcggactt caataacgtg cccaaaatgc gggcactgcg
aggttgggaa ccgggatggg 2040gaggcttttc agtgccttag ttgcggcaaa
acgtgcaatg ccgaccttga cgtggctacc 2100cataatctga ctcaagtcgc
ccttacagga aaaacaatgc cgaaacgcga ggaacctaga 2160gatgcccagg
gcacagctcc agcccgaaaa acaaagaagg cgtcaaagag caaggctccg
2220ccagccgaac gagaggacca aactccagca caggaaccgt cccagacttc
cggaagcgga 2280cccaagaaaa aacgcaaggt ggaagatcct aagaaaaagc
ggaaagtgag cctgggcagc 2340ggctccgatt acaaagatga cgatgacaaa
gactacaagg atgatgatga taagggatcc 2400ggcgcaacaa acttctctct
gctgaaacaa gccggagatg tcgaagagaa tcctggaccg 2460accgagtaca
agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
2520accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
tccggaccgc 2580cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
cgcgcgtcgg gctcgacatc 2640ggcaaggtgt gggtcgcgga cgacggcgcc
gcggtggcgg tctggaccac gccggagagc 2700gtcgaagcgg gggcggtgtt
cgccgagatc ggcccgcgca tggccgagtt gagcggttcc 2760cggctggccg
cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
2820gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg
tctgggcagc 2880gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
gggtgcccgc cttcctggag 2940acctccgcgc cccgcaacct ccccttctac
gagcggctcg gcttcaccgt caccgccgac 3000gtcgaggtgc ccgaaggacc
gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaacg 3060cgttaagaat
tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat
3120ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
cccactgtcc 3180tttcctaata aaatgaggaa attgcatcgc attgtctgag
taggtgtcat tctattctgg 3240ggggtggggt ggggcaggac agcaaggggg
aggattggga agagaatagc aggcatgctg 3300gggagcggcc gcaggaaccc
ctagtgatgg agttggccac tccctctctg cgcgctcgct 3360cgctcactga
ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
3420cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
tttctcctta 3480cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
ccatagtacg cgccctgtag 3540cggcgcatta agcgcggcgg gtgtggtggt
tacgcgcagc gtgaccgcta cacttgccag 3600cgccttagcg cccgctcctt
tcgctttctt cccttccttt ctcgccacgt tcgccggctt 3660tccccgtcaa
gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
3720cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
cgccctgata 3780gacggttttt cgccctttga cgttggagtc cacgttcttt
aatagtggac tcttgttcca 3840aactggaaca acactcaact ctatctcggg
ctattctttt gatttataag ggattttgcc 3900gatttcggtc tattggttaa
aaaatgagct gatttaacaa aaatttaacg cgaattttaa 3960caaaatatta
acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
4020atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
gggcttgtct 4080gctcccggca tccgcttaca gacaagctgt gaccgtctcc
gggagctgca tgtgtcagag 4140gttttcaccg tcatcaccga aacgcgcgag
acgaaagggc ctcgtgatac gcctattttt 4200ataggttaat gtcatgataa
taatggtttc ttagacgtca ggtggcactt ttcggggaaa 4260tgtgcgcgga
acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
4320gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
tgagtattca 4380acatttccgt gtcgccctta ttcccttttt tgcggcattt
tgccttcctg tttttgctca 4440cccagaaacg ctggtgaaag taaaagatgc
tgaagatcag ttgggtgcac gagtgggtta 4500catcgaactg gatctcaaca
gcggtaagat ccttgagagt tttcgccccg aagaacgttt 4560tccaatgatg
agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
4620cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
ttgagtactc 4680accagtcaca gaaaagcatc ttacggatgg catgacagta
agagaattat gcagtgctgc 4740cataaccatg agtgataaca ctgcggccaa
cttacttctg acaacgatcg gaggaccgaa 4800ggagctaacc gcttttttgc
acaacatggg ggatcatgta actcgccttg atcgttggga 4860accggagctg
aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
4920ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
cccggcaaca 4980attaatagac tggatggagg cggataaagt tgcaggacca
cttctgcgct cggcccttcc 5040ggctggctgg tttattgctg ataaatctgg
agccggtgag cgtggaagcc gcggtatcat 5100tgcagcactg gggccagatg
gtaagccctc ccgtatcgta gttatctaca cgacggggag 5160tcaggcaact
atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
5220gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
taaaacttca 5280tttttaattt aaaaggatct aggtgaagat cctttttgat
aatctcatga ccaaaatccc 5340ttaacgtgag ttttcgttcc actgagcgtc
agaccccgta gaaaagatca aaggatcttc 5400ttgagatcct ttttttctgc
gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 5460agcggtggtt
tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
5520cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
gccaccactt 5580caagaactct gtagcaccgc ctacatacct cgctctgcta
atcctgttac cagtggctgc 5640tgccagtggc gataagtcgt gtcttaccgg
gttggactca agacgatagt taccggataa 5700ggcgcagcgg tcgggctgaa
cggggggttc gtgcacacag cccagcttgg agcgaacgac 5760ctacaccgaa
ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
5820gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
gcacgaggga 5880gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
gggtttcgcc acctctgact 5940tgagcgtcga tttttgtgat gctcgtcagg
ggggcggagc ctatggaaaa acgccagcaa 6000cgcggccttt ttacggttcc
tggccttttg ctggcctttt gctcacatgt gagggcctat 6060ttcccatgat
tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
6120ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
aagtaataat 6180ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
ggactatcat atgcttaccg 6240taacttgaaa gtatttcgat ttcttggctt
tatatatctt gtggaaagga cgaaacaccg 6300gtcggaacgc tcaacgattg
cccctcacga ggggacagaa gagctaatgc tcttcatttt 6360ttttggtacc
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
6420cccgcccatt gacgtcaata gtaacgccaa tagggacttt ccattgacgt
caatgggtgg 6480agtatttacg gtaaactgcc cacttggcag tacatcaagt
gtatcatatg ccaagtacgc 6540cccctattga cgtcaatgac ggtaaatggc
ccgcctggca ttgtgcccag tacatgacct 6600tatgggactt tcctacttgg
cagtacatct acgtattagt catcgctatt accatggtcg 6660aggtgagccc
cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
6720tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
gggggggggc 6780gcgcgccagg cggggcgggg sggggsgrgg ggsggggsgg
ggsgrggcgg agaggtgcgg 6840cggcagccaa tcagagcggc gcgctccgaa
agtttccttt tatggcgagg cggcggcggc 6900ggcggcccta taaaaagcga
agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc 6960ccgtgccccg
ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
7020tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
ctgagcaaga 7080ggtaagggtt taagggatgg ttggttggtg gggtattaat
gtttaattac ctggagcacc 7140tgcctgaaat cacttttttt caggttggac
cggtgccacc 71801627207DNAArtificial SequenceSynthetic sequence
162atggaaaaag aaataactga gctcaccaag attaggcgcg agtttccgaa
taaaaagttc 60agcagcactg atatgaagaa ggcaggtaag ttgttgaagg cagaaggtcc
tgatgctgtt 120agagacttcc tgaactcctg ccaggagatt atcggggatt
ttaagccgcc tgtaaagaca 180aacatagtca gcatatcacg accctttgag
gagtggcctg ttagtatggt ggggcgcgcc 240atccaggaat attactttag
tttgacaaaa gaggaattgg agtccgtcca tcccggaact 300tccagcgagg
atcacaagtc cttctttaac ataactggcc tgagcaatta caattatacg
360tcagtccaag gcttgaatct catcttcaaa aatgcgaagg ccatatacga
cgggactctg 420gttaaagcaa acaataaaaa taagaagttg gaaaaaaagt
tcaatgagat taaccacaag 480cgaagccttg aggggcttcc tataattacg
ccggatttcg aggaaccctt tgatgagaat 540ggccatctga ataatccgcc
aggtattaat cgaaatattt acggctacca aggatgtgcc 600gctaaagtat
tcgttccttc caagcataaa atggtatccc tccctaaaga atacgaaggg
660tacaaccggg atccgaacct gtccttggcg ggcttccgaa atcggctcga
gataccggag 720ggggagcccg gtcacgtgcc atggtttcag cgcatggata
tcccggaagg ccagatcggg 780cacgtaaata agattcaacg attcaatttc
gttcatggca agaattcagg aaaagtcaaa 840ttcagcgata agacaggacg
ggtaaaacgc taccatcatt ccaagtataa agatgccact 900aagccttaca
aatttcttga agaatccaag aaagtcagtg ctctggactc catccttgcc
960attatcacaa tcggtgatga ctgggtagtg tttgacattc gcggtctgta
tagaaatgtt 1020ttttatcgcg aactggcaca gaagggcctg acagcagtgc
agctgctgga tctgtttacg 1080ggggatccgg tgattgaccc gaagaagggc
gttgtgacat tcagctataa ggaaggcgtg 1140gttccagtat tttcacagaa
gatcgttcca aggttcaaga gtcgagacac gctcgagaaa 1200ttgaccagtc
aaggacctgt ggcgctgctc tcagtcgacc tcggccaaaa tgaaccagtg
1260gcggcaaggg tttgtagctt gaagaacata aatgataaga tcacattgga
taattcttgc 1320agaatctcct tcctggatga ctacaaaaaa caaatcaaag
actacagaga ttccctggac 1380gaacttgaaa tcaagatacg actggaagca
atcaattctc tggaaactaa ccaacaagta 1440gaaattcgcg acctggatgt
attcagtgct gatcgggcaa aggcaaacac tgtagatatg 1500ttcgacatcg
acccaaattt gatatcctgg gattcaatga gcgacgcgag ggtgagcacg
1560caaataagcg atctttatct gaagaatggg ggtgacgaat ctcgagtata
tttcgaaatt 1620aacaacaaac ggataaagcg atctgattat aacattagtc
agctggtgag gccaaagctt 1680tccgacagca ctcggaagaa tctgaacgat
tctatatgga agttgaaaag aactagtgaa 1740gaatatttga aattgtccaa
acgaaagttg gaactgagca gagctgttgt gaactacact 1800atccgccaga
gcaagctcct ctccggaatt aacgacattg ttataatact tgaggacctg
1860gatgtaaaaa aaaaattcaa tggcaggggc attcgagata tcggatggga
caacttcttc 1920agctccagga aagagaacag gtggttcatt ccggcattcc
ataaggcttt ctcagagctt 1980tcaagcaacc ggggcctctg tgtcatcgaa
gtcaacccgg catggacatc tgccacctgt 2040cccgactgcg ggttctgtag
taaagagaac agagatggca ttaattttac ctgtcgcaag 2100tgcggtgtct
cttaccacgc ggacatagat gttgccactc ttaatatagc ccgggtggcc
2160gttctcggca agcctatgtc cggacccgcc gaccgcgaga gactgggcga
tactaagaaa 2220ccccgggtag caaggagccg aaagactatg aaacggaaag
atattagcaa tagcaccgtt 2280gaggctatgg ttacagccgg aagcggaccc
aagaaaaaac gcaaggtgga agatcctaag 2340aaaaagcgga aagtgagcct
gggcagcggc tccgattaca aagatgacga tgacaaagac 2400tacaaggatg
atgatgataa gggatccggc gcaacaaact tctctctgct gaaacaagcc
2460ggagatgtcg aagagaatcc tggaccgacc gagtacaagc ccacggtgcg
cctcgccacc 2520cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
cgttcgccga ctaccccgcc 2580acgcgccaca ccgtcgatcc ggaccgccac
atcgagcggg tcaccgagct gcaagaactc 2640ttcctcacgc gcgtcgggct
cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg 2700gtggcggtct
ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
2760ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
ggaaggcctc 2820ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
ccaccgtcgg agtctcgccc 2880gaccaccagg gcaagggtct gggcagcgcc
gtcgtgctcc ccggagtgga ggcggccgag 2940cgcgccgggg tgcccgcctt
cctggagacc tccgcgcccc gcaacctccc cttctacgag 3000cggctcggct
tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
3060atgacccgca agcccggtgc ctgaacgcgt taagaattcc tagagctcgc
tgatcagcct 3120cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
ctcccccgtg ccttccttga 3180ccctggaagg tgccactccc actgtccttt
cctaataaaa tgaggaaatt gcatcgcatt 3240gtctgagtag gtgtcattct
attctggggg gtggggtggg gcaggacagc aagggggagg 3300attgggaaga
gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt
3360tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
aaggtcgccc 3420gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
agcgcgcagc tgcctgcagg 3480ggcgcctgat gcggtatttt ctccttacgc
atctgtgcgg tatttcacac cgcatacgtc 3540aaagcaacca tagtacgcgc
cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 3600gcgcagcgtg
accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc
3660ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
ggctcccttt 3720agggttccga tttagtgctt tacggcacct cgaccccaaa
aaacttgatt tgggtgatgg 3780ttcacgtagt gggccatcgc cctgatagac
ggtttttcgc cctttgacgt tggagtccac 3840gttctttaat agtggactct
tgttccaaac tggaacaaca ctcaactcta tctcgggcta 3900ttcttttgat
ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat
3960ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
tatggtgcac 4020tctcagtaca atctgctctg atgccgcata gttaagccag
ccccgacacc cgccaacacc 4080cgctgacgcg ccctgacggg cttgtctgct
cccggcatcc gcttacagac aagctgtgac 4140cgtctccggg agctgcatgt
gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg 4200aaagggcctc
gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
4260gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
tatttttcta 4320aatacattca aatatgtatc cgctcatgag acaataaccc
tgataaatgc ttcaataata 4380ttgaaaaagg aagagtatga gtattcaaca
tttccgtgtc gcccttattc ccttttttgc 4440ggcattttgc cttcctgttt
ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 4500agatcagttg
ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
4560tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
ttctgctatg 4620tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
ctcggtcgcc gcatacacta 4680ttctcagaat gacttggttg agtactcacc
agtcacagaa aagcatctta cggatggcat 4740gacagtaaga gaattatgca
gtgctgccat aaccatgagt gataacactg cggccaactt 4800acttctgaca
acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
4860tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
caaacgacga 4920gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
cgcaaactat taactggcga 4980actacttact ctagcttccc ggcaacaatt
aatagactgg atggaggcgg ataaagttgc 5040aggaccactt ctgcgctcgg
cccttccggc tggctggttt attgctgata aatctggagc 5100cggtgagcgt
ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg
5160tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
atagacagat 5220cgctgagata ggtgcctcac tgattaagca ttggtaactg
tcagaccaag tttactcata 5280tatactttag attgatttaa aacttcattt
ttaatttaaa aggatctagg tgaagatcct 5340ttttgataat ctcatgacca
aaatccctta acgtgagttt tcgttccact gagcgtcaga 5400ccccgtagaa
aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
5460cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
aagagctacc 5520aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
ataccaaata ctgttcttct 5580agtgtagccg tagttaggcc accacttcaa
gaactctgta gcaccgccta catacctcgc 5640tctgctaatc ctgttaccag
tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 5700ggactcaaga
cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
5760cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
agcgtgagct 5820atgagaaagc gccacgcttc ccgaagggag aaaggcggac
aggtatccgg taagcggcag 5880ggtcggaaca ggagagcgca cgagggagct
tccaggggga aacgcctggt atctttatag 5940tcctgtcggg tttcgccacc
tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 6000gcggagccta
tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
6060gccttttgct cacatgtgag ggcctatttc ccatgattcc ttcatatttg
catatacgat 6120acaaggctgt tagagagata attggaatta atttgactgt
aaacacaaag atattagtac 6180aaaatacgtg acgtagaaag taataatttc
ttgggtagtt tgcagtttta aaattatgtt 6240ttaaaatgga ctatcatatg
cttaccgtaa cttgaaagta tttcgatttc ttggctttat 6300atatcttgtg
gaaaggacga aacaccgacc aaaacgacta ttgattgccc agtacgctgg
6360gacagaagag ctaatgctct tcattttttt tggtacccgt tacataactt
acggtaaatg 6420gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
gtcaatagta acgccaatag 6480ggactttcca ttgacgtcaa tgggtggagt
atttacggta aactgcccac ttggcagtac 6540atcaagtgta tcatatgcca
agtacgcccc ctattgacgt caatgacggt aaatggcccg 6600cctggcattg
tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
6660tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc
actctcccca 6720tctccccccc ctccccaccc ccaattttgt atttatttat
tttttaatta ttttgtgcag 6780cgatgggggc gggggggggg ggggggcgcg
cgccaggcgg ggcggggsgg ggsgrggggs 6840ggggsggggs grggcggaga
ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt 6900ttccttttat
ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
6960cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc
tcgcgccgcc 7020cgccccggct ctgactgacc gcgttactcc cacaggtgag
cgggcgggac ggcccttctc 7080ctccgggctg taattagctg agcaagaggt
aagggtttaa gggatggttg gttggtgggg 7140tattaatgtt taattacctg
gagcacctgc ctgaaatcac tttttttcag gttggaccgg 7200tgccacc
720716328DNAArtificial SequenceSynthetic sequence 163gttaactgcc
gcataggcag cttagaaa 2816428DNAArtificial SequenceSynthetic sequence
164gtgaaccgcc gtataggcag cttagaaa 2816536RNAArtificial
SequenceSynthetic sequencemisc_feature(4)..(4)y is c or
umisc_feature(6)..(6)r is a or gmisc_feature(7)..(7)d is a, g, or
umisc_feature(10)..(10)w is a or umisc_feature(12)..(12)h is a, c,
or umisc_feature(13)..(13)y is c or umisc_feature(15)..(15)r is a
or
gmisc_feature(22)..(22)r is a or gmisc_feature(23)..(23)d is a, g
or umisc_feature(24)..(24)w is a or umisc_feature(25)..(26)r is a
or gmisc_feature(27)..(27)n is a, c, g, or umisc_feature(28)..(28)k
is g or umisc_feature(29)..(29)d is a, g, or
umisc_feature(32)..(32)k is g or umisc_feature(33)..(33)n is a, c,
g, or umisc_feature(34)..(34)d is a, g, or umisc_feature(35)..(35)r
is a or gmisc_feature(36)..(36)b is c, g, or u 165gucycrdcgw
ahygrgcaau crdwrrnkdu ukndrb 3616636RNAArtificial SequenceSynthetic
sequence 166gucccaacga auugggcaau caaaaaggau uggauc
3616736RNAArtificial SequenceSynthetic sequence 167gucucagcgu
acugagcaau caaaagguuu cgcagg 3616836RNAArtificial SequenceSynthetic
sequence 168gucucgacua aucgagcaau cguuugagau cucucc
3616936RNAArtificial SequenceSynthetic sequence 169guccccucgu
gaggggcaau cguugagcgu uccgac 3617036RNAArtificial SequenceSynthetic
sequence 170gucccagcgu acugggcaau caauagucgu uuuggu
3617136RNAArtificial SequenceSynthetic sequence 171gucgcggcgu
accgcgcaau gagagucugu ugccau 3617236RNAArtificial SequenceSynthetic
sequence 172gucuccucgu aaggagcaau cuauuagucu ugaaag
3617336RNAArtificial SequenceSynthetic sequence 173gucucggcgc
accgagcaau cagcgagguc uucuac 3617436RNAArtificial SequenceSynthetic
sequencemisc_feature(1)..(1)v is a, c, or gmisc_feature(2)..(2)y is
c or umisc_feature(3)..(3)h is a, c, or umisc_feature(4)..(4)n is
a, c, g, or umisc_feature(5)..(5)m is a or cmisc_feature(8)..(8)h
is a, c, or umisc_feature(9)..(9)m is a or cmisc_feature(10)..(10)n
is a, c, g, or umisc_feature(11)..(12)y is c or
umisc_feature(13)..(13)w is a or umisc_feature(13)..(13)w is a or
umisc_feature(14)..(14)h is a, c, or umisc_feature(15)..(15)y is c
or umisc_feature(21)..(21)y is c or umisc_feature(24)..(24)r is a
or gmisc_feature(25)..(25)d is a, g or umisc_feature(27)..(27)w is
a or umisc_feature(30)..(30)h is a, c, or umisc_feature(31)..(31)y
is c or umisc_feature(33)..(33)r is a or g 174vyhnmaahmn yywhygauug
cycrduwcgh ygrgac 3617536RNAArtificial SequenceSynthetic sequence
175gauccaaucc uuuuugauug cccaauucgu ugggac 3617636RNAArtificial
SequenceSynthetic sequence 176ccugcgaaac cuuuugauug cucaguacgc
ugagac 3617736RNAArtificial SequenceSynthetic sequence
177ggagagaucu caaacgauug cucgauuagu cgagac 3617836RNAArtificial
SequenceSynthetic sequence 178gucggaacgc ucaacgauug ccccucacga
ggggac 3617936RNAArtificial SequenceSynthetic sequence
179accaaaacga cuauugauug cccaguacgc ugggac 3618036RNAArtificial
SequenceSynthetic sequence 180auggcaacag acucucauug cgcgguacgc
cgcgac 3618136RNAArtificial SequenceSynthetic sequence
181cuuucaagac uaauagauug cuccuuacga ggagac 3618236RNAArtificial
SequenceSynthetic sequence 182guagaagacc ucgcugauug cucggugcgc
cgagac 3618349RNAArtificial SequenceSynthetic sequence
183caacgauugc cccuacagag gggacagcug guaaugggau accuugugc
4918436RNAArtificial SequenceSynthetic sequence 184ugccccuaca
gaggggacag cugguaaugg gauacc 3618529DNAArtificial SequenceSynthetic
sequence 185caattcgacc attaccctat ggaacacga 2918629DNAArtificial
SequenceSynthetic sequence 186gttaagctgg taatgggata ccttgtgct
2918736RNAArtificial SequenceSynthetic sequence 187ugcucgauua
gucgagacag cugguaaugg gauacc 3618829DNAArtificial SequenceSynthetic
sequence 188caattcgacc attaccctat ggaacacga 2918928DNAArtificial
SequenceSynthetic sequence 189gttagctggt aatgggatac cttgtgct
2819036RNAArtificial SequenceSynthetic sequence 190ugccccuaca
gaggggacag cugguaaugg gauacc 3619129DNAArtificial SequenceSynthetic
sequence 191caattcgacc attaccctat ggaacacga 2919229DNAArtificial
SequenceSynthetic sequence 192gttaagctgg taatgggata ccttgtgct
2919336RNAArtificial SequenceSynthetic sequence 193ugcccaguac
gcugggacag cugguaaugg gauacc 3619429DNAArtificial SequenceSynthetic
sequence 194taagtcgacc attaccctat ggaacacga 2919529DNAArtificial
SequenceSynthetic sequence 195attcagctgg taatgggata ccttgtgct
2919660RNAArtificial SequenceSynthetic sequence 196cacaggagag
aucucaaacg auugcucgau uagucgagac agcugguaau gggauaccuu
6019760RNAArtificial SequenceSynthetic sequence 197uaaugucgga
acgcucaacg auugccccua cagaggggac ugccgccucc gcgacgccca
6019835DNAArtificial SequenceSynthetic sequence 198ctggagttgt
cccaattctt gttgaattag atggt 3519935DNAArtificial SequenceSynthetic
sequence 199aacatttccg tgtcgccctt attccctttt ttgcg
3520020DNAArtificial SequenceSynthetic sequence 200ggcgagggcg
atgccaccta 2020120DNAArtificial SequenceSynthetic sequence
201ttcaagtccg ccatgcccga 2020220DNAArtificial SequenceSynthetic
sequence 202ggtgaaccgc atcgagctga 2020320DNAArtificial
SequenceSynthetic sequence 203cttgtacagc tcgtccatgc
2020420DNAArtificial SequenceSynthetic sequence 204tcgggcagca
gcacggggcc 2020520DNAArtificial SequenceSynthetic sequence
205tagttgtact ccagcttgtg 2020620DNAArtificial SequenceSynthetic
sequence 206tggccgttta cgtcgccgtc 2020720DNAArtificial
SequenceSynthetic sequence 207aagaagtcgt gctgcttcat
2020820DNAArtificial SequenceSynthetic sequence 208accggggtgg
tgcccatcct 2020920DNAArtificial SequenceSynthetic sequence
209agcgtgtccg gcgagggcga 2021020DNAArtificial SequenceSynthetic
sequence 210atctgcacca ccggcaagct 2021120DNAArtificial
SequenceSynthetic sequence 211gagggcgaca ccctggtgaa
2021220DNAArtificial SequenceSynthetic sequence 212accagggtgt
cgccctcgaa 2021320DNAArtificial SequenceSynthetic sequence
213ttctgcttgt cggccatgat 2021420DNAArtificial SequenceSynthetic
sequence 214accttgatgc cgttcttctg 2021520DNAArtificial
SequenceSynthetic sequence 215tgctggtagt ggtcggcgag
2021620DNAArtificial SequenceSynthetic sequence 216gtgaccgccg
ccgggatcac 2021720DNAArtificial SequenceSynthetic sequence
217gggtctttgc tcagcttgga 2021820DNAArtificial SequenceSynthetic
sequence 218tggcggatct tgaagttcac 2021920DNAArtificial
SequenceSynthetic sequence 219tggctgttgt agttgtactc
2022020DNAArtificial SequenceSynthetic sequence 220tactccagct
tgtgccccag 2022120DNAArtificial SequenceSynthetic sequence
221ccgtcctcct tgaagtcgat 2022220DNAArtificial SequenceSynthetic
sequence 222ccgtcgtcct tgaagaagat 2022320DNAArtificial
SequenceSynthetic sequence 223ccgtaggtgg catcgccctc
2022420DNAArtificial SequenceSynthetic sequence 224ccggtggtgc
agatgaactt 2022520DNAArtificial SequenceSynthetic sequence
225aagaagatgg tgcgctcctg 2022620DNAArtificial SequenceSynthetic
sequence 226cgtgatggtc tcgattgagt 2022760RNAArtificial
SequenceSynthetic sequence 227cacaggagag aucucaaacg auugcucgau
uagucgagac agcugguaau gggauaccuu 6022860RNAArtificial
SequenceSynthetic sequence 228uaaugucgga acgcucaacg auugccccuc
acgaggggac ugccgccucc gcgacgccca 6022960RNAArtificial
SequenceSynthetic sequence 229auuaaccaaa acgacuauug auugcccagu
acgcugggac uaugagcuua uguacaucaa 6023052RNAArtificial
SequenceSynthetic sequence 230gaccuuuuua auuucuacuc uuguagauaa
agugcucauc auuggaaaac gu 522311906DNAArtificial SequenceSynthetic
sequence 231ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
catccatagt 60tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
ctggccccag 120tgctgcaatg ataccgcggg acccacgctc accggctcca
gatttatcag caataaacca 180gccagccgga agggccgagc gcagaagtgg
tcctgcaact ttatccgcct ccatccagtc 240tattaattgt tgccgggaag
ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 300tgttgccatt
gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
360ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
aaaaagcggt 420tagctccttc ggtcctccga tcgttgtcag aagtaagttg
gccgcagtgt tatcactcat 480ggttatggca gcactgcata attctcttac
tgtcatgcca tccgtaagat gcttttctgt 540gactggtgag tactcaacca
agtcattctg agaatagtgt atgcggcgac cgagttgctc 600ttgcccggcg
tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
660cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
tgagatccag 720ttcgatgtaa cccactcgtg cacccaactg atcttcagca
tcttttactt tcaccagcgt 780ttctgggtga gcaaaaacag gaaggcaaaa
tgccgcaaaa aagggaataa gggcgacacg 840gaaatgttga atactcatac
tcttcctttt tcaatattat tgaagcattt atcagggtta 900ttgtctcatg
agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
960gcgcacattt ccccgaaaag tgccacctgt catgaccaaa atcccttaac
gtgagttttc 1020gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
tcttcttgag atcctttttt 1080tctgcgcgta atctgctgct tgcaaacaaa
aaaaccaccg ctaccagcgg tggtttgttt 1140gccggatcaa gagctaccaa
ctctttttcc gaaggtaact ggcttcagca gagcgcagat 1200accaaatact
gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
1260accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
gtggcgataa 1320gtcgtgtctt accgggttgg actcaagacg atagttaccg
gataaggcgc agcggtcggg 1380ctgaacgggg ggttcgtgca cacagcccag
cttggagcga acgacctaca ccgaactgag 1440atacctacag cgtgagctat
gagaaagcgc cacgcttccc gaagggagaa aggcggacag 1500gtatccggta
agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
1560cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
gtcgattttt 1620gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
agcaacgcgg cctttttacg 1680gttcctggcc ttttgctggc cttttgctca
catgttcttt cctgcgttat cccctgattc 1740tgtggataac cgtgcggccg
ccccttgtag ttaagctggt aatgggatac cttgtgctac 1800agcggccgcg
attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
1860ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagtta
19062321898DNAArtificial SequenceSynthetic sequence 232gctcttgccc
ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 60tcatcattgg
aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
120ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
actttcacca 180gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
aaaaaaggga ataagggcga 240cacggaaatg ttgaatactc atactcttcc
tttttcaata ttattgaagc atttatcagg 300gttattgtct catgagcgga
tacatatttg aatgtattta gaaaaataaa caaatagggg 360ttccgcgcac
atttccccga aaagtgccac ctgtcatgac caaaatccct taacgtgagt
420tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
tgagatcctt 480tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
accgctacca gcggtggttt 540gtttgccgga tcaagagcta ccaactcttt
ttccgaaggt aactggcttc agcagagcgc 600agataccaaa tactgttctt
ctagtgtagc cgtagttagg ccaccacttc aagaactctg 660tagcaccgcc
tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
720ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
gcgcagcggt 780cgggctgaac ggggggttcg tgcacacagc ccagcttgga
gcgaacgacc tacaccgaac 840tgagatacct acagcgtgag ctatgagaaa
gcgccacgct tcccgaaggg agaaaggcgg 900acaggtatcc ggtaagcggc
agggtcggaa caggagagcg cacgagggag cttccagggg 960gaaacgcctg
gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
1020ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
gcggcctttt 1080tacggttcct ggccttttgc tggccttttg ctcacatgtt
ctttcctgcg ttatcccctg 1140attctgtgga taaccgtgcg gccgcccctt
gtagttaagc tggtaatggg ataccttgtg 1200ctacagcggc cgcgattatc
aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 1260agttttaaat
caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
1320atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
tgcctgactc 1380cccgtcgtgt agataactac gatacgggag ggcttaccat
ctggccccag tgctgcaatg 1440ataccgcggg acccacgctc accggctcca
gatttatcag caataaacca gccagccgga 1500agggccgagc gcagaagtgg
tcctgcaact ttatccgcct ccatccagtc tattaattgt 1560tgccgggaag
ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
1620gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
ctccggttcc 1680caacgatcaa ggcgagttac atgatccccc atgttgtgca
aaaaagcggt tagctccttc 1740ggtcctccga tcgttgtcag aagtaagttg
gccgcagtgt tatcactcat ggttatggca 1800gcactgcata attctcttac
tgtcatgcca tccgtaagat gcttttctgt gactggtgag 1860tactcaacca
agtcattctg agaatagtgt atgcggcg 18982331898DNAArtificial
SequenceSynthetic sequence 233gctcttgccc ggcgtcaata cgggataata
ccgcgccaca tagcagaact ttaaaagtgc 60tcatcattgg aaaacgttct tcggggcgaa
aactctcaag gatcttaccg ctgttgagat 120ccagttcgat gtaacccact
cgtgcaccca actgatcttc agcatctttt actttcacca 180gcgtttctgg
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
240cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
atttatcagg 300gttattgtct catgagcgga tacatatttg aatgtattta
gaaaaataaa caaatagggg 360ttccgcgcac atttccccga aaagtgccac
ctgtcatgac caaaatccct taacgtgagt 420tttcgttcca ctgagcgtca
gaccccgtag aaaagatcaa aggatcttct tgagatcctt 480tttttctgcg
cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
540gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
agcagagcgc 600agataccaaa tactgttctt ctagtgtagc cgtagttagg
ccaccacttc aagaactctg 660tagcaccgcc tacatacctc gctctgctaa
tcctgttacc agtggctgct gccagtggcg 720ataagtcgtg tcttaccggg
ttggactcaa gacgatagtt accggataag gcgcagcggt 780cgggctgaac
ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
840tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
agaaaggcgg 900acaggtatcc ggtaagcggc agggtcggaa caggagagcg
cacgagggag cttccagggg 960gaaacgcctg gtatctttat agtcctgtcg
ggtttcgcca cctctgactt gagcgtcgat 1020ttttgtgatg ctcgtcaggg
gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 1080tacggttcct
ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
1140attctgtgga taaccgtgcg gccgcccctt gtagccaagc tggtaatggg
ataccttgtg 1200ctacagcggc cgcgattatc aaaaaggatc ttcacctaga
tccttttaaa ttaaaaatga 1260agttttaaat caatctaaag tatatatgag
taaacttggt ctgacagtta ccaatgctta 1320atcagtgagg cacctatctc
agcgatctgt ctatttcgtt catccatagt tgcctgactc 1380cccgtcgtgt
agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
1440ataccgcggg acccacgctc accggctcca gatttatcag caataaacca
gccagccgga 1500agggccgagc gcagaagtgg tcctgcaact ttatccgcct
ccatccagtc tattaattgt 1560tgccgggaag ctagagtaag tagttcgcca
gttaatagtt tgcgcaacgt tgttgccatt 1620gctacaggca tcgtggtgtc
acgctcgtcg tttggtatgg cttcattcag ctccggttcc 1680caacgatcaa
ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
1740ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
ggttatggca 1800gcactgcata attctcttac tgtcatgcca tccgtaagat
gcttttctgt gactggtgag 1860tactcaacca agtcattctg agaatagtgt atgcggcg
189823456DNAArtificial SequenceSynthetic sequence 234cggccgcccc
ttgtagttaa gctggtaatg ggataccttg tgctacagcg gccgcg
5623556DNAArtificial SequenceSynthetic sequence 235cgcggccgct
gtagcacaag gtatcccatt accagcttaa ctacaagggg cggccg
5623656DNAArtificial SequenceSynthetic sequence 236cggccgcccc
ttgtaattca gctggtaatg ggataccttg tgctacagcg gccgcg
5623756DNAArtificial SequenceSynthetic sequence 237cgcggccgct
gtagcacaag gtatcccatt accagctgaa ttacaagggg cggccg
5623841RNAArtificial SequenceSynthetic sequence 238cgcuguagca
caagguaucc cauuaccagc uuaacuacaa g 4123948DNAArtificial
SequenceSynthetic sequence 239gtggccgttt aaaagtgctc atcattggaa
aacgtaggat gggcacca 4824032RNAArtificial SequenceSynthetic sequence
240aguauuuaau cguugcaaga ggcgcugcgu uu 3224125RNAArtificial
SequenceSynthetic sequence 241caacgauugc cccucacgag gggac
2524237RNAArtificial SequenceSynthetic sequence 242caacgauugc
cccucacgag gggacagcug guaaugg 3724339RNAArtificial
SequenceSynthetic sequence 243caacgauugc cccucacgag gggacagcug
guaauggga 3924441RNAArtificial SequenceSynthetic sequence
244caacgauugc cccucacgag gggacagcug guaaugggau a
4124543RNAArtificial SequenceSynthetic sequence 245caacgauugc
cccucacgag gggacagcug guaaugggau acc 4324645RNAArtificial
SequenceSynthetic sequence 246caacgauugc cccucacgag gggacagcug
guaaugggau accuu 4524747RNAArtificial SequenceSynthetic sequence
247caacgauugc cccucacgag gggacagcug guaaugggau accuugu
4724849RNAArtificial SequenceSynthetic sequence 248caacgauugc
cccucacgag gggacagcug guaaugggau accuugugc 4924943RNAArtificial
SequenceSynthetic sequence 249aaacgauugc ucgauuaguc gagacagcug
guaaugggau acc
4325043RNAArtificial SequenceSynthetic sequence 250uauugauugc
ccaguacgcu gggacagcug guaaugggau acc 43
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