U.S. patent application number 16/748267 was filed with the patent office on 2020-06-04 for method for fragmenting genomic dna using cas9.
The applicant listed for this patent is AGILENT TECHNOLOGIES, INC.. Invention is credited to Robert A. Ach, Bram Herman, Brian Jon Peter, Michael Walter.
Application Number | 20200172959 16/748267 |
Document ID | / |
Family ID | 51985517 |
Filed Date | 2020-06-04 |
United States Patent
Application |
20200172959 |
Kind Code |
A1 |
Peter; Brian Jon ; et
al. |
June 4, 2020 |
Method for Fragmenting Genomic DNA Using CAS9
Abstract
A method for fragmenting a genome is provided. In certain
embodiments, the method comprises: (a) combining a genomic sample
containing genomic DNA with a plurality of Cas9-gRNA complexes,
wherein the Cas9-gRNA complexes comprise a Cas9 protein and a set
of at least 10 Cas9-associated guide RNAs that are complementary to
different, pre-defined, sites in a genome, to produce a reaction
mixture; and (b) incubating the reaction mixture to produce at
least 5 fragments of the genomic DNA. Also provided is a
composition comprising at least 100 Cas9-associated guide RNAs that
are each complementary to a different, pre-defined, site in a
genome. Kits for performing the method are also provided. In
addition, other methods, compositions and kits for manipulating
nucleic acids are also provided.
Inventors: |
Peter; Brian Jon; (Los
Altos, CA) ; Ach; Robert A.; (San Francisco, IL)
; Walter; Michael; (Blaubeuren, DE) ; Herman;
Bram; (Letchworth Garden City, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AGILENT TECHNOLOGIES, INC. |
Santa Clara |
CA |
US |
|
|
Family ID: |
51985517 |
Appl. No.: |
16/748267 |
Filed: |
January 21, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15339510 |
Oct 31, 2016 |
10577644 |
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16748267 |
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14290896 |
May 29, 2014 |
9873907 |
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15339510 |
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61828507 |
May 29, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y10T 436/143333
20150115; C12Q 1/6869 20130101; C12N 15/1003 20130101; C12Y 301/00
20130101; C12Q 1/6806 20130101; C12N 15/1034 20130101; C12N 9/22
20130101 |
International
Class: |
C12Q 1/6806 20060101
C12Q001/6806; C12N 9/22 20060101 C12N009/22; C12Q 1/6869 20060101
C12Q001/6869; C12N 15/10 20060101 C12N015/10 |
Claims
1. A composition comprising: a set of at least 10 Cas9-associated
guide RNAs that are each complementary to a different, pre-defined
sequence.
2. The composition of claim 1, wherein each sequence is a genomic
sequence.
3. The composition of claim 1, wherein each sequence is a human
genomic sequence.
4. The composition of claim 1, wherein each sequence is a genomic
sequence from a different pathogen.
5. The composition of claim 1, further comprising a Cas9
nuclease.
6. The composition of claim 1, wherein said RNAs are in
solution.
7. The composition of claim 1, wherein said RNAs are tethered to a
substrate in an array.
Description
CROSS-REFERENCING
[0001] This application is a divisional of U.S. application Ser.
No. 15/339,510, filed Oct. 31, 2016, which is a continuation of
U.S. application Ser. No. 14/290,896, filed May 29, 2014, now U.S.
Pat. No. 9,873,907, which claims the benefit of U.S. provisional
application Ser. Nos. 61/828,507, filed May 29, 2013 and 61/831,061
filed Jun. 4, 2013, all of which are incorporated by reference
herein.
BACKGROUND
[0002] Methods for fragmenting a genome find use in a variety of
genomic analysis applications, including, but not limited to SNP
analysis, sequencing, mutation detection and the detection of
chromosomal rearrangements.
SUMMARY
[0003] The present disclosure provides, inter alia, a method for
fragmenting a genome. In certain embodiments, the method comprises:
(a) combining a genomic sample containing genomic DNA with a
plurality of Cas9-gRNA complexes, wherein the Cas9-gRNA complexes
comprise a Cas9 protein and a set of at least 10 Cas9-associated
guide RNAs that are complementary to different, pre-defined, sites
in a genome, to produce a reaction mixture; and (b) incubating the
reaction mixture to produce at least 5 fragments of the genomic
DNA. Also provided is a composition comprising at least 100
Cas9-associated guide RNAs that are each complementary to a
different, pre-defined, site in a genome. Kits for performing the
method are also provided. Further provided are other methods,
compositions and kits for manipulating nucleic acids.
BRIEF DESCRIPTION OF THE FIGURES
[0004] FIG. 1 schematically illustrates a method for fragmenting a
genome.
DEFINITIONS
[0005] The term "sample" as used herein relates to a material or
mixture of materials, typically, although not necessarily, in
liquid form, containing one or more analytes of interest. A sample
may have a complexity of least 10.sup.3, at least 10.sup.4, at
least 10.sup.5, 10.sup.6 or 10.sup.7 or more.
[0006] The term "nucleic acid sample," as used herein denotes a
sample containing nucleic acids.
[0007] The term "nucleotide" is intended to include those moieties
that contain not only the known purine and pyrimidine bases, but
also other heterocyclic bases that have been modified. Such
modifications include methylated purines or pyrimidines, acylated
purines or pyrimidines, alkylated riboses or other heterocycles. In
addition, the term "nucleotide" includes those moieties that
contain hapten or fluorescent labels and may contain not only
conventional ribose and deoxyribose sugars, but other sugars as
well. Modified nucleosides or nucleotides also include
modifications on the sugar moiety, e.g., wherein one or more of the
hydroxyl groups are replaced with halogen atoms or aliphatic
groups, or are functionalized as ethers, amines, or the like.
[0008] The term "nucleic acid" and "polynucleotide" are used
interchangeably herein to describe a polymer of any length, e.g.,
greater than about 2 bases, greater than about 10 bases, greater
than about 100 bases, greater than about 500 bases, greater than
1000 bases, up to about 10,000 or more bases composed of
nucleotides, e.g., deoxyribonucleotides or ribonucleotides, and may
be produced enzymatically or synthetically (e.g., PNA as described
in U.S. Pat. No. 5,948,902 and the references cited therein) which
can hybridize with naturally occurring nucleic acids in a sequence
specific manner analogous to that of two naturally occurring
nucleic acids, e.g., can participate in Watson-Crick base pairing
interactions. Naturally-occurring nucleotides include guanine,
cytosine, adenine and thymine (G, C, A and T, respectively).
[0009] The term "target polynucleotide," as used herein, refers to
a polynucleotide of interest under study. In certain embodiments, a
target polynucleotide contains one or more sequences that are of
interest and under study.
[0010] The term "oligonucleotide" as used herein denotes a
single-stranded multimer of nucleotide of from about 2 to 200
nucleotides, up to 500 nucleotides in length. Oligonucleotides may
be synthetic or may be made enzymatically, and, in some
embodiments, are 10 to 50 nucleotides in length. Oligonucleotides
may contain ribonucleotide monomers (i.e., may be
oligoribonucleotides) or deoxyribonucleotide monomers. An
oligonucleotide may be 10 to 20, 21 to 30, 31 to 40, 41 to 50,
51-60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, or
200 to 250 nucleotides in length, for example.
[0011] The terms "double stranded" and "duplex" as used herein,
describes two complementary polynucleotides that are base-paired,
i.e., hybridized together.
[0012] The term "amplifying" as used herein refers to generating
one or more copies of a target nucleic acid, using the target
nucleic acid as a template.
[0013] The terms "determining," "measuring," "evaluating,"
"assessing," "assaying," and "analyzing" are used interchangeably
herein to refer to any form of measurement, and include determining
if an element is present or not. These terms include both
quantitative and/or qualitative determinations. Assessing may be
relative or absolute. "Assessing the presence of" includes
determining the amount of something present, as well as determining
whether it is present or absent.
[0014] The term "using" has its conventional meaning, and, as such,
means employing, e.g., putting into service, a method or
composition to attain an end. For example, if a program is used to
create a file, a program is executed to make a file, the file
usually being the output of the program. In another example, if a
computer file is used, it is usually accessed, read, and the
information stored in the file employed to attain an end. Similarly
if a unique identifier, e.g., a barcode is used, the unique
identifier is usually read to identify, for example, an object or
file associated with the unique identifier.
[0015] As used herein, the term "single nucleotide polymorphism,"
or "SNP" for short, refers to single nucleotide position in a
genomic sequence for which two or more alternative alleles are
present at appreciable frequency (e.g., at least 1%) in a
population.
[0016] The term "free in solution," as used here, describes a
molecule, such as a polynucleotide, that is not bound or tethered
to another molecule.
[0017] The term "partitioning," with respect to a genome, refers to
the separation of one part of the genome from the remainder of the
genome to produce a product that is isolated from the remainder of
the genome. The term "partitioning" encompasses enriching.
[0018] The term "genomic region," as used herein, refers to a
region of a genome, e.g., an animal or plant genome such as the
genome of a microbe (e.g., a bacterium), human, monkey, rat, fish
or insect or plant. In certain cases, an oligonucleotide used in
the method described herein may be designed using a reference
genomic region, i.e., a genomic region of known nucleotide
sequence, e.g., a chromosomal region whose sequence is deposited at
NCBI's Genbank database or other databases, for example. Such an
oligonucleotide may be employed in an assay that uses a sample
containing a test genome, where the test genome contains a cleaving
site for a nicking endonuclease adjacent to a binding site for the
oligonucleotide. The precise nucleotide sequence that flanks the
oligonucleotide binding site in a test genome may be known or
unknown.
[0019] The term "affinity tag," as used herein, refers to moiety
that can be used to separate a molecule to which the affinity tag
is attached from other molecules that do not contain the affinity
tag. In certain cases, an "affinity tag" may bind to the "capture
agent," where the affinity tag specifically binds to the capture
agent, thereby facilitating the separation of the molecule to which
the affinity tag is attached from other molecules that do not
contain the affinity tag. Examples of affinity tags include biotin,
digoxygenin, peptide tags, and protein tags (e.g., his-tags and the
like).
[0020] As used herein, the term "biotin moiety" refers to an
affinity agent that includes biotin or a biotin analogue such as
desthiobiotin, oxybiotin, 2'-iminobiotin, diaminobiotin, biotin
sulfoxide, biocytin, etc. Biotin moieties bind to streptavidin with
an affinity of at least 10.sup.-8 M. A biotin affinity agent may
also include a linker, e.g., -LC-biotin, -LC-LC-Biotin, -SLC-Biotin
or -PEG.sub.n-Biotin where n is 3-12.
[0021] A "plurality" contains at least 2 members. In certain cases,
a plurality may have at least 10, at least 100, at least 100, at
least 10,000, at least 100,000, at least 10.sup.6, at least
10.sup.7, at least 10.sup.8 or at least 10.sup.9 or more
members.
[0022] The term "adaptor-ligated," as used herein, refers to a
nucleic acid that has been ligated to an adaptor. The adaptor can
be ligated to a 5' end and/or a 3' end of a nucleic acid
molecule.
[0023] The term "adaptor" refers to a nucleic acid that is
ligatable to one or both strands of a double-stranded DNA molecule.
In some embodiments, an adaptor may be a hairpin adaptor. In
another embodiment, an adaptor may itself be composed of two
distinct oligonucleotide molecules that are base paired with one
another. As would be apparent, a ligatable end of an adaptor may be
designed to compatible with overhangs made by cleavage by a
restriction enzyme, or it may have blunt ends.
[0024] The term "genotyping," as used herein, refers to any type of
analysis of a nucleic acid sequence, and includes sequencing,
polymorphism (SNP) analysis, and analysis to identify
rearrangements.
[0025] The term "sequencing," as used herein, refers to a method by
which the identity of at least 10 consecutive nucleotides (e.g.,
the identity of at least 20, at least 50, at least 100 or at least
200 or more consecutive nucleotides) of a polynucleotide are
obtained.
[0026] The term "next-generation sequencing" refers to the
so-called parallelized sequencing-by-synthesis or
sequencing-by-ligation platforms currently employed by Illumina,
Life Technologies, and Roche etc. Next-generation sequencing
methods may also include nanopore sequencing methods or
electronic-detection based methods such as Ion Torrent technology
commercialized by Life Technologies.
[0027] The term "target sequence" refers to a sequence in a
double-stranded DNA molecule, where the target sequence is bound,
and, optionally cleaved or nicked by Cas9. In many cases, a target
sequence may be unique in any one starting molecule and, as will be
described in greater detail below, multiple different starting
molecules (e.g., overlapping fragments) may contain the same target
sequence. In some cases, the target sequence may be degenerate,
that is, the target sequence may have base positions that may have
variable bases. These positions may be denoted as Y, R, N, etc.,
where Y and R denote pyrimidine and purine bases, respectively, and
N denotes any of the 4 bases.
[0028] The term "cleaving," as used herein, refers to a reaction
that breaks the phosphodiester bonds between two adjacent
nucleotides in both strands of a double-stranded DNA molecule,
thereby resulting in a double-stranded break in the DNA molecule.
.
[0029] The term "nicking," as used herein, refers to a reaction
that breaks the phosphodiester bond between two nucleotides in one
strand of a double-stranded DNA molecule to produce a 3' hydroxyl
group and a 5' phosphate group.
[0030] The terms "cleavage site," and "nick site," as used herein,
refers to the site at which a double-stranded DNA molecule has been
cleaved or nicked.
[0031] The term "Cas9-associated guide RNA" refers to a guide RNA
as described above (comprising a crRNA molecule and a tracrRNA
molecule, or comprising an RNA molecule that includes both crRNA
and tracrRNA sequences). The Cas9-associated guide RNA may exist as
isolated RNA, or as part of a Cas9-gRNA complex.
[0032] Reference to a Cas9-associated guide RNA is "complementary
to" another sequence is not intended to mean that the entire guide
RNA is complementary to the other sequence. A Cas9-associated guide
RNA that is complementary to another sequence comprises a sequence
that is complementary to the other sequence. Specifically, it is
known that a Cas9 complex can specifically bind to a target
sequence that has as few as 8 or 9 bases of complementarity with
the guide Cas9-associated guide RNA in the complex. Off site
binding can be decreased by increasing the length of
complementarity, e.g., to 15 or 20 bases.
[0033] The terms "Cas9 enzyme" and "Cas9-gRNA complex" refer to a
complex comprising a Cas9 protein and a guide RNA (gRNA). The guide
RNA may be composed of two molecules, i.e., one RNA ("crRNA") which
hybridizes to a target and provides sequence specificity, and one
RNA, the "tracrRNA", which is capable of hybridizing to the crRNA.
Alternatively, the guide RNA may be a single molecule (i.e., a
sgRNA) that contains crRNA and tracrRNA sequences. A Cas9 protein
may be at least 60% identical (e.g., at least 70%, at least 80%, or
90% identical, at least 95% identical or at least 98% identical or
at least 99% identical) to a wild type Cas9 protein, e.g., to the
Streptococcus pyogenes Cas9 protein. The Cas9 protein may have all
the functions of a wild type Cas 9 protein, or only one or some of
the functions, including binding activity, nuclease activity, and
nuclease activity.
[0034] For Cas9 to successfully bind to DNA, the target sequence in
the genomic DNA should be complementary to the gRNA sequence and
must be immediately followed by the correct protospacer adjacent
motif or "PAM" sequence. The PAM sequence is present in the DNA
target sequence but not in the gRNA sequence. Any DNA sequence with
the correct target sequence followed by the PAM sequence will be
bound by Cas9. The PAM sequence varies by the species of the
bacteria from which Cas9 was derived. The most widely used Type II
CRISPR system is derived from S. pyogenes and the PAM sequence is
NGG located on the immediate 3' end of the gRNA recognition
sequence. The PAM sequences of Type II CRISPR systems from
exemplary bacterial species include: Streptococcus pyogenes (NGG),
Neisseria meningitidis (NNNNGATT), Streptococcus thermophilus
(NNAGAA) and Treponema denticola (NAAAAC).
[0035] The term "Cas9 nickase" referes to a modified version of the
Cas9-gRNA complex, as described above, containing a single inactive
catalytic domain, i.e., either the RuvC- or the HNH-domain. With
only one active nuclease domain, the Cas9 nickase cuts only one
strand of the target DNA, creating a single-strand break or "nick".
A Cas9 nickase is still able to bind DNA based on gRNA specificity,
though nickases will only cut one of the DNA strands. The majority
of CRISPR plasmids currently being used are derived from S.
pyogenes and the RuvC domain can be inactivated by an amino acid
substitution at position D10 (e.g., D10A) and the HNH domain can be
inactivated by an by an amino acid substitution at position H840
(e.g., H840A), or at positions corresponding to those amino acids
in other proteins. As is known, the D10 and H840 variants of Cas9
cleave a Cas9-induced bubble at specific sites on opposite strands
of the DNA. Depending on which mutant is used, the guide
RNA-hybridized strand or the non-hybridized strand may be
cleaved.
[0036] The term "mutant Cas9 protein that has inactivated nuclease
activity" refers to a Cas protein that has inactivated HNH and RuvC
nucleases. Such a protein can bind to a target site in
double-stranded DNA (where the target site is determined by the
guide RNA), but the protein is unable to cleave or nick the
double-stranded DNA.
[0037] As used herein and unless indicated to the contrary, the
term "Cas9-fragment complex" refers to a complex containing a
Cas9-gRNA and a DNA fragment to which the Cas9-gRNA complex
binds.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0038] Before the present invention is described in greater detail,
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.
[0039] 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.
[0040] 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.
[0041] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not 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.
[0042] 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. 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.
[0043] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
[0044] The following references are explicitly incorporated by
reference for their teachings on Cas9, gRNA, and other reagents
that can be used herein: Gasiunas et al (Proc. Natl. Acad. Sci.
2012 109: E2579-E2586), Karvelis et al (Biochem. Soc. Trans. 2013
41:1401-6), Pattanayak et al (Nat. Biotechnol. 2013 31: 839-43),
Jinek et al. (Elife 2013 2: e00471), Jiang et al (Nat. Biotechnol.
2013 31:233-9), Hwang et al (Nat. Biotechnol. 2013 31: 227-9), Mali
et al (Science 2013 339:823-6), Cong et al (Science. 2013 339:
819-23), DiCarlo et al (Nucleic Acids Res. 2013 41: 4336-43) and Qi
et al (Cell. 2013 152: 1173-83).
[0045] As would be appreciated, the method described below may be
employed to fragment a wide variety of different types of DNA,
including plasmids, cDNA and genomic DNA.
Method for Fragmenting Genomic DNA
[0046] As noted above, a method for fragmenting a genome is
provided. In certain embodiments, the method comprises: (a)
combining a genomic sample containing genomic DNA with a plurality
of Cas9-gRNA complexes, wherein the Cas9-gRNA complexes comprise a
Cas9 protein and a set of at least 10 Cas9-associated guide RNAs
that are complementary to different, pre-defined, sites in a
genome, to produce a reaction mixture; and (b) incubating the
reaction mixture to produce at least 5 fragments of the genomic
DNA.
[0047] As would be apparent, this reaction may be done in vitro,
i.e., in a cell-free environment using isolated genomic DNA. The
method may be used to isolate double-stranded DNA fragments from
virtually any source, including but not limited to total genomic
DNA and complementary DNA (cDNA), plasmid DNA, mitochondrial DNA,
synthetic DNA, and BAC clones, etc. Furthermore, any organism,
organic material or nucleic acid-containing substance can be used
as a source of nucleic acids to be processed in accordance with the
present method including, but not limited to, plants, animals
(e.g., reptiles, mammals, insects, worms, fish, etc.), tissue
samples, bacteria, fungi (e.g., yeast), phage, viruses, cadaveric
tissue, archaeological/ancient samples, etc. In certain
embodiments, the genomic DNA used in the method may be derived from
a mammal, wherein certain embodiments the mammal is a human. In the
description set forth above and below the method is used to
fragment genomic DNA. However, it is recognized that the same
method can be used to fragment DNA from other sources, e.g.,
cDNA.
[0048] The genomic DNA may be isolated from any organism. The
organism may be a prokaryote or a eukaryote. In certain cases, the
organism may be a plant, e.g., Arabidopsis or maize, or an animal,
including reptiles, mammals, birds, fish, and amphibians. In some
cases, the test genome may be human or rodent, such as a mouse or a
rat. Methods of preparing genomic DNA for analysis is routine and
known in the art, such as those described by Ausubel, F. M. et al.,
(Short protocols in molecular biology, 3rd ed., 1995, John Wiley
& Sons, Inc., New York) and Sambrook, J. et al. (Molecular
cloning: A laboratory manual, 2.sup.nd ed., 1989, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.). In certain
cases, the sample used may contain total genomic DNA, which may be
unamplified or amplified, e.g., genomic DNA that has been amplified
by a whole genome amplification method, that may or may not be
already fragmented by other means, e.g., fragmented into fragments
that are over 10 kb, or over 50 kb in length.
[0049] The guide RNAs used in the method may be designed so that
they direct binding of the Cas9-gRNA complexes to pre-determined
cleavage sites in a genome. In certain cases, the cleavage sites
may be chosen so as to release a fragment that contains a region of
unknown sequence, or a region containing a SNP, nucleotide
insertion, nucleotide deletion, rearrangement, etc. Since genomic
isolation methods, and the nucleotide sequences of many organisms
(including many bacteria, fungi, plants and animals, e.g., mammals
such as human, primates, and rodents such as mouse and rat) are
known, designing guide RNAs for use in the present method should be
within the skill of one of skilled in the art.
[0050] Cas9-gRNA complexes can be programmed to bind to any
sequence, provided that the sequence has a PAM motif. In theory,
the Cas9-gRNA complexes could cleave the genomic DNA to produce
fragments in the range of 30-50 bp. However, in practice, the
minimal interval between the cleavage sites may be e.g., in the
range of 50-200 bp.
[0051] The Cas9-gRNA complexes may comprise a set of at least 10,
at least 100, at least 1,000, at least 10,000, at least 50,000 or
at least 100,000 or more different Cas9-associated guide RNAs that
are each complementary to a different, pre-defined, site in a
genome. The distance between neighboring sites may vary greatly
depending on the desired application. In some embodiments, the
distance between neighboring sites is in the range of 1 kb to 200
kb, and, in particular embodiments, the sites may be chosen to
release fragments of a similar, defined, size, e.g., where at least
95% of the predicted fragments have a size that is within 20%,
within 10% or within 5% of a chosen size, where the chosen size is
in the range of 1 kb to 10 kb or 10 kb to 100 kb, for example. In
certain cases, cleavage sites for the Cas9-gRNA complexes may be
chosen to release fragments that are of a size suitable for cloning
into a particular vector, e.g., a cosmid, fosmid, bac or
bacteriophage, that only accepts fragments of a particular
size.
[0052] In these embodiments, fosmid clones have been useful for
genomic analysis due to their restricted length (.about.37 to 42
kb) and ability to preserve human sequence in E. coli without
extensive rearrangement. However, a disadvantage of fosmid cloning
approaches is the inability to target desired sequences. In this
embodiment, one can design custom Cas9/CRISPR nucleases programmed
with guide RNAs that target the enzyme to the ends of a set of
approximately 40-kilobase fragments. After cleavage of the genome
with the Cas9/CRISPR nucleases, the fraction of total DNA in the
size range of 40 kb would be enriched for the target segments. This
DNA could be cloned into fosmid vectors and the resulting library
would be enriched for 40 kilobase target sequences, suitable for
long-read sequencing. Careful preparation of genomic DNA with an
average length above 50 kb (e.g., using specialized kits such as
Qiagen's Genomic Tips or the MegaLong kit from G Biosciences) will
improve the efficiency of recovering target fragments and will
decrease the fraction of off-target sequences.
[0053] The method may be used to produce at least 10, at least 100,
at least 1,000, at least 10,000, at least 50,000 or at least
100,000 or more fragments of a genome. Depending on how the method
is implemented, the fragments may be distributed throughout the
genome, or they may be distributed in one or more specific regions
of a genome (e.g., 1, at least 10, at least 50, at least 100 or at
least 1,000 or more specific regions), where each region may be
cleaved to produce multiple fragments.
[0054] In some cases, the fragments produced by the method may be
cloned into a vector, e.g., a fosmid, bac or cosmid vector for
storage and later analysis. In some cases, the fragments may be
treated with Taq polymerase to produce that contain a 3' A
overhang, and then cloned by TA cloning, The fragments (whether or
not they are cloned in a vector) may be genotyped, e.g., sequenced.
In some cases, the fragments may be amplified prior to cloning
and/or analysis, which may involve ligating adaptors onto the ends
of the fragments, and amplifying the fragments using primers that
hybridize to the ligated adaptors.
[0055] In particular embodiments, the fragments may be sequenced.
In certain embodiments, the fragment may be amplified using primers
that are compatible with use in, e.g., Illumina's reversible
terminator method, Roche's pyrosequencing method (454), Life
Technologies' sequencing by ligation (the SOLiD platform) or Life
Technologies' Ion Torrent platform. Examples of such methods are
described in the following references: Margulies et al (Nature 2005
437: 376-80); Ronaghi et al (Analytical Biochemistry 1996 242:
84-9); Shendure et al (Science 2005 309: 1728-32); Imelfort et al
(Brief Bioinform. 2009 10:609-18); Fox et al (Methods Mol Biol.
2009;553:79-108); Appleby et al (Methods Mol Biol. 2009;513:19-39)
and Morozova et al (Genomics. 2008 92:255-64), which are
incorporated by reference for the general descriptions of the
methods and the particular steps of the methods, including all
starting products, reagents, and final products for each of the
steps. In some cases, the fragments may be subjected to target
enrichment methods prior to sequencing. Target enrichment methods
are known in the art and encompass methods such as SureSelect and
HaloPlex technologies commercialized by Agilent Technologies,
PCR-amplification based strategies, and the like.
[0056] In one embodiment, the fragments may be sequenced using
nanopore sequencing (e.g. as described in Soni et al. 2007 Clin
Chem 53: 1996-2001, or as described by Oxford Nanopore
Technologies). Nanopore sequencing is a single-molecule sequencing
technology whereby a single molecule of DNA is sequenced directly
as it passes through a nanopore. A nanopore is a small hole, of the
order of 1 nanometer in diameter. Immersion of a nanopore in a
conducting fluid and application of a potential (voltage) across it
results in a slight electrical current due to conduction of ions
through the nanopore. The amount of current which flows is
sensitive to the size and shape of the nanopore. As a DNA molecule
passes through a nanopore, each nucleotide on the DNA molecule
obstructs the nanopore to a different degree, changing the
magnitude of the current through the nanopore in different degrees.
Thus, this change in the current as the DNA molecule passes through
the nanopore represents a reading of the DNA sequence. Nanopore
sequencing technology is disclosed in U.S. Pat. Nos. 5,795,782,
6,015,714, 6,627,067, 7,238,485 and 7,258,838 and U.S. Pat Appln
Nos. 2006003171 and 20090029477.
[0057] Additionally, the reaction mixture may also comprise a set
of Cas9-associated guide RNAs that are complementary to repetitive
sequences in the genomic DNA, and the incubating results in
cleavage of the repetitive sequences. This method may used to
effectively remove repetitive sequence from a sample, where the
term "repetitive sequence" refers to a segment of DNA containing a
sequence of nucleotides that is repeated for at least 5, 10, 15,
20, 30, 40, 50, 60, 80, or 100 or more times. Repetitive sequences
can include single nucleotide repeats (homopolymer stretches, e.g.,
poly A or poly T tails), di-nucleotide repeats (e.g., ATAT or
AGAG), tri-nucleotide repeats, tetranucleotide repeats, telomeric
repetitive elements and the like. Repetitive sequences also
include, but not limited to, ALU, LINE (long interspersed genetic
elements, which are non-coding), SINE (short interspersed genetic
elements, which also are non-coding), and certain transposons such
as L and P element sequences. ALU elements are a type of SINE
element, roughly 300 base pairs in length. In certain embodiments,
the repeat sequences may be cleaved to any suitable size, e.g., to
a size in the range of 13-200 bases and each repeat may be cleaved
at multiple sites. In some cases, the repetitive sequences are
cleaved into fragments that range in size from 20 bases to 1 kb.
The smaller fragments (the repetitive sequences) may be separated
from the larger fragments (containing the non-repetitive sequences)
by any suitable method, including by size exclusion. The longer
fragments can be processed (e.g., cloned, amplified, sequenced,
etc.). In some embodiments, certain longer fragments may be
selected by target enrichment prior to further analysis. Such
methods are known in the art, for example, methods described in
U.S. Pat. No. 8,017,328 and US patent application US20130323725,
which are incorporated herein by reference.
[0058] In certain embodiments, inhibitors of DNases can be used to
reduce degradation of DNA. DNase inhibitors that are compatible
with Cas9 include, but are not limited to, 2-mercaptoethanol and
actin.
[0059] In some embodiments, the method may further comprise
analyzing the genome after cleavage of one or more repetitive
sequences.
[0060] In some embodiments, the method may comprise enriching a
fraction of the genome after cleavage of one or more repetitive
sequences.
Compositions
[0061] In addition to the method described above, a number of
compositions are also provided. In certain embodiments, the
composition may contain a set of at least 10 Cas9-associated guide
RNAs that are each complementary to a different, pre-defined, site
in a genome. The composition may comprise, e.g., at least 10, at
least 15, at least 20, at least 30, at least 50, at least 75, at
least 100, at least 200, at least 300, at least 400, at least 500,
at least 600, at least 700, at least 800, at least 900, at least
1,000, or at least 10,000 or more guide RNAs. The sites to which
the Cas9-associated guide RNAs bind are immediately downstream from
a PAM trinucleotide (e.g., CCN). The guide RNAs may be in solution,
or they may be in dried form, e.g., lyophilized. The guide RNAs may
be at least 20, at least 30, at least 50, at least 75, at least
100, at least 150, at least 180, at least 200, at least 220, at
least 240, or at least 260 nucleotides long. Such compositions may
be employed in any embodiment disclosed herein.
[0062] As would be apparent, the composition may additionally
contain a single Cas9 protein. The composition may also contain
genomic DNA, e.g., microbial or mammalian genomic DNA such as human
genomic DNA.
[0063] The guide RNAs may be synthesized on a solid support in an
array, where the oligonucleotides are grown in situ.
Oligonucleotide arrays can be fabricated using any means, including
drop deposition from pulse jets or from fluid-filled tips, etc., or
using photolithographic means. Polynucleotide precursor units (such
as nucleotide monomers), in the case of in situ fabrication can be
deposited. Oligonucleotides synthesized on a solid support may then
be cleaved off to generate the population of oligonucleotides. Such
methods are described in detail in, for example U.S. Pat. Nos.
7,385,050, 6,222,030, 6,323,043, and US Pat Appln Pub No.
2002/0058802, etc., the disclosures of which are incorporated
herein by reference. The oligonucleotides may be tethered to a
solid support via a cleavable linker, and cleaved from the support
before use.
[0064] In some embodiments, the Cas9-associated guide RNAs are each
specific for a different, pre-defined, site in genomic DNA.
[0065] In some embodiments, the Cas9-associated guide RNAs are each
specific for a different, pre-defined, site in mammalian genomic
DNA.
[0066] In some embodiments, the Cas9-associated guide RNAs that are
each specific for a different, pre-defined, site in human genomic
DNA.
[0067] In some embodiments, the Cas9-associated guide RNAs are each
specific for a different, pre-defined, site in microbial genomic
DNA.
[0068] In some embodiments, the composition comprises one or a
plurality of Cas9-associated guide RNA binding to the genome of one
pathogen and one or a plurality of Cas9-associated guide RNA
binding to the genome of another pathogen.
[0069] In some embodiments, the sites to which the Cas9-associated
guide RNAs bind are spaced along the genomic DNA at a defined
interval.
[0070] In some embodiments, the defined interval is in the range of
1 kb to 100 kb.
[0071] In some embodiments, the composition further comprises a set
of Cas9-associated guide RNAs that are capable of binding
repetitive sequences in genomic DNA.
[0072] In some embodiments, the composition further comprises a
Cas9 nuclease.
[0073] In some embodiments, the Cas9-associated guide RNAs are in
solution as a mixture.
[0074] In some embodiments, the Cas9-associated guide RNAs are
tethered to a substrate in an array. In some embodiments, the
composition comprises a DNase inhibitor.
Kits
[0075] Also provided by the subject invention are kits for
practicing the subject method, as described above. The subject kit
contains mutant Cas9 protein and set of at least 2, at least 5, at
least 10, at least 15, at least 20, at least 30, at least 50, at
least 75, at least 100, at least 200, at least 300, at least 400,
at least 500, at least 600, at least 700, at least 800, at least
900, at least 1,000, or at least 10,000 or more guide RNAs, as
described above. The guide RNAs may in the form of a dried pellet
or an aqueous solution. . The guide RNAs may be at least 20, at
least 30, at least 50, at least 75, at least 100, at least 150, at
least 180, at least 200, at least 220, at least 240, or at least
260 nucleotides long.
[0076] In addition to the instructions, the kits may also include
one or more control genomes and or oligonucleotides for use in
testing the kit. The subject kit may further include instructions
for using the components of the kit to practice the subject
methods. The instructions for practicing the subject methods are
generally recorded on a suitable recording medium. For example, the
instructions may be printed on a substrate, such as paper or
plastic, etc. As such, the instructions may be present in the kit
as a package insert, in the labeling of the container of the kit or
components thereof (i.e., associated with the packaging or
subpackaging), etc. In other embodiments, the instructions are
present as an electronic storage data file present on a suitable
computer readable storage medium, e.g. CD-ROM, diskette, etc. In
yet other embodiments, the actual instructions are not present in
the kit, but means for obtaining the instructions from a remote
source, e.g., via the internet, are provided. An example of this
embodiment is a kit that includes a web address where the
instructions can be viewed and/or from which the instructions can
be downloaded. As with the instructions, this means for obtaining
the instructions is recorded on a suitable substrate.
[0077] The various components of the kit may be in separate
containers, where the containers may be contained within a single
housing, e.g., a box.
[0078] In some embodiments, the Cas9-associated guide RNAs are each
specific for a different, pre-defined, site in genomic DNA.
[0079] In some embodiments, the Cas9-associated guide RNAs are each
specific for a different, pre-defined, site in mammalian genomic
DNA.
[0080] In some embodiments, the Cas9-associated guide RNAs that are
each specific for a different, pre-defined, site in human genomic
DNA.
[0081] In some embodiments, the Cas9-associated guide RNAs are each
specific for a different, pre-defined, site in microbial genomic
DNA.
[0082] In some embodiments, the kit comprises one or a plurality of
Cas9-associated guide RNA binding to the genome of one pathogen and
one or a plurality of Cas9-associated guide RNA binding to the
genome of another pathogen.
[0083] In some embodiments, the sites to which the Cas9-associated
guide RNAs bind are spaced along the genomic DNA at a defined
interval.
[0084] In some embodiments, the defined interval is in the range of
1 kb to 100 kb.
[0085] In some embodiments, the kit further comprises a set of
Cas9-associated guide RNAs that are capable of binding repetitive
sequences in genomic DNA.
[0086] In some embodiments, the kit further comprises a Cas9
nuclease.
[0087] In some embodiments, the Cas9-associated guide RNAs are in
solution as a mixture.
[0088] In some embodiments, the Cas9-associated guide RNAs are
tethered to a substrate in an array. In some embodiments, the kit
comprises a DNase inhibitor.
Utility
[0089] The above-described method may be used to fragment a genome
in a defined way, i.e., to produce fragments of one or more chosen
regions of a genome. The fragments produced by the subject method
may be arbitrarily chosen or, in some embodiments, may have a
common function, structure or expression. While the above-described
method is not so limited, the method may be employed to isolate
promoters, terminators, exons, introns, entire genes, homologous
genes, sets of gene sequences that are linked by function,
expression or sequence, regions containing insertion, deletion or
translocation breakpoints or SNP-containing regions, for example.
Alternatively, the method could be used to reduce the sequence
complexity of a genome prior to analysis, or to enrich for genomic
regions of interest.
[0090] In certain embodiments the method may be used to produce
fragments of interest (i.e., one or more regions of a genome),
where the resultant sample is at least 50% free, e.g., at least 80%
free, at least 90% free, at least 95% free, at least 99% free of
the other parts of the genome. In particular embodiments, the
products of the method may be amplified before analysis. In other
embodiments, the products of the method may be analyzed in an
unmodified form, i.e., without amplification.
[0091] As noted above, the method may be employed to isolate a
region of interest from a genome. The isolated region may be
analyzed by any analysis method including, but not limited to, DNA
sequencing (using Sanger, pyrosequencing or the sequencing systems
of Roche/454, Helicos, Illumina/Solexa, and ABI (SOLiD)), a
polymerase chain reaction assay, a hybridization assay, a
hybridization assay employing a probe complementary to a mutation,
a microarray assay, a bead array assay, a primer extension assay,
an enzyme mismatch cleavage assay, a branched hybridization assay,
a NASBA assay, a molecular beacon assay, a cycling probe assay, a
ligase chain reaction assay, an invasive cleavage structure assay,
an ARMS assay, or a sandwich hybridization assay, for example. Some
products (e.g., single-stranded products) produced by the method
may be sequenced, and analyzed for the presence of SNPs or other
differences relative to a reference sequence. As would be clear to
one skilled in the art, the proposed method may be useful in
several fields of genetic analysis, by allowing the artisan to
focus his or her analysis on a genomic region of interest.
[0092] The subject method finds particular use in SNP haplotyping
of a chromosomal region that contains two or more SNPs, for
enriching for DNA sequences for paired-end sequencing methods, for
generating target fragments for long-read sequences, isolating
inversion, deletion, and translocation breakpoints, for sequencing
entire gene regions (exons and introns) to uncover mutations
causing aberrant splicing or regulation, and for the production of
long probes for chromosome imaging, e.g., Bionanomatrix, optical
mapping, or fiber-FISH-based methods.
[0093] In particular cases, the method described above can also be
used for long-range haplotyping by using hemizygous deletions to
differentially label maternal and paternal chromosomes. The method
may be employed to capture such hemizygous sequences together with
adjoining sequence. In this way, maternal and paternal copies of
DNA could be separated and analyzed independently. This would
enable haplotype phased sequencing.
[0094] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
ALTERNATIVE EMBODIMENTS
Cas9-Transposase Fusion Proteins
[0095] A fusion protein comprising a Cas9 protein and a transposase
is provided.
[0096] In some embodiments, the Cas9 protein has inactivated
nuclease activity
[0097] In some embodiments, the Cas9 protein is fused to the
N-terminus of the transposase.
[0098] In some embodiments, the Cas9 protein is fused to the
C-terminus of the transposase.
[0099] A complex comprising a fusion protein comprising a Cas9
protein and a transposase is provided, where the complex further
comprises a Cas9-associated guide RNA and a transposon.
[0100] In some embodiments, Cas9 protein and Cas9-associated guide
RNA directs the transposon to a defined site in a genome, thereby
allowing the transposase to insert the transposable element at a
defined site.
[0101] In some embodiments, the transposon comprises one or more of
a primer binding site, a molecular barcode or a promoter.
[0102] In some embodiments, the promoter is a .PHI.29 promoter.
[0103] Also provided is a method comprising contacting the complex
with a genome, thereby causing the transposable element to be
inserted into the genome proximal at a site to which the Cas9
protein binds.
[0104] In some embodiments, the method may be done by contacting a
plurality of complexes with a genome, wherein each complex
comprises a different guide RNA, and the different guide RNAs are
complementary to defined sites in the genome, and inserting a
plurality of transposons into the genome.
[0105] In some embodiments, the sites to which the guide RNAs bind
are distributed across a target sequence at defined intervals
(e.g., in the range of 100 bp to 5 kb), thereby producing
transposon insertions at defined intervals.
[0106] In some embodiments, the sequences between the transposon
insertions are amplified using PCR primers that bind to primer
binding sites in the transposon insertions.
[0107] In some embodiments, the transposon is biotinylated.
[0108] In some embodiments, the transposase is a Sleeping Beauty,
Piggybac or Tn5 transposase.
[0109] Transposases are enzymes derived from transposable elements
that randomly break DNA and insert a transposable-element DNA that
encodes the transposase. Transposases have been used in genetic and
molecular biology applications to rapidly integrate DNA "tags" into
a target sample of DNA (usually genomic DNA) as part of an
insertional mutagenesis screen (in vivo) or more recently to create
next-generation sequencing libraries (in vitro).
[0110] As with transposable element integration, the integration of
DNA tags show little sequence bias except insertion between TA
dinucleotides (which are duplicated during transposition and flank
the integration site). For some next generation sequencing (NGS)
applications applications, whole-genome surveys are benefitted by
the random integration events garnered from transposition, which is
the basis of the Nextera whole-genome library preparation
technology from Illumina. However, for creating targeted NGS
libraries, it would be advantageous to target a transposase to
specific genomic locations to enable the rapid production of
"directed" NGS libraries. The types of "targeted" NGS libraries
envisioned here would obviate the hybridization-based selection
approaches used in target capture protocols, as these selections
take extra time, and could permit time-sensitive applications (such
as diagnostics).
[0111] In this embodiment, Cas9/crRNA is used to provide an
efficient targeting system to place desired sequences (e.g.,
barcodes, promoters, primer binding sites, etc.) in specific
locations on target DNAs. In some embodiments, mutant Cas9 protein
that has inactivated nuclease activity may targeted to a specific
genomic locus for the purposes of: (a) rapidly producing a targeted
next generation sequencing (NGS) library preparation suitable for
diagnostic or research purposes via Cas9-targeted transposition of
NGS adaptors or (b) Cas9-targeted transposition of a promoter,
e.g., a minimal D29 origin of replication that will enable the
isothermal amplification of >50 kb of "tagged" genomic DNAs,
which can be used for amplification, isolation and detection of
chromosomal rearrangements from tumor samples or the interrogation
of unknown regions of microbial DNAs. The mutant Cas9 protein is
inactivated in the sense that it can bind, but it cannot cleave,
the sequence to which it has been programmed to bind by the gRNA
complexed to it. In certain embodiments, the Cas9 protein has amino
acid substitutions at D10 and H840, or sites corresponding thereto.
In particular embodiments, the Cas9 protein may have D10A and H840A
substitutions (or equivalent substitutions at positions
corresponding to D10 and H840 in the Streptococcus Cas9
protein).
[0112] In one embodiment, a
cas9.sup..DELTA.HNH/.DELTA.RuvC::transposase (such as ISY100)
fusion protein loaded with a transposon containing, e.g.,
next-generation sequencing (NGS) adaptors or common sequences that
could be primed to generate NGS libraries by PCR, are produced. In
this example, targeting the cas9-transposase fusion protein to
specific gDNA loci will enable the targeted one-step integration of
NGS adaptors at specific genomic sites, thus an NGS library that is
ready for sequencing can be produced rapidly and without
hybrid-selection approaches. An added feature could be the use of
biotinylated DNA "tags" such that these targeted integrants may be
separated from the gDNA that is not part of the NGS library.
[0113] A related approach could involve tethering wildtype
(cleavage-competent) Cas9 to a topoisomerase linked to an NGS
adaptor or other nucleic acid sequence. After dsDNA target
cleavage, the topoisomerase can ligate the tethered NGS adaptor to
the blunt ends generated by Cas9 cleavage and produce a similar
outcome as (a) above.
[0114] Alternatively, the NGS adaptor in the method described above
can be replaced with a minimal origin of replication for B.
subtilis phage .PHI.29. This will result in the deposition of a
.PHI.29 replication origin at a Cas9-directed genomic site. These
replication origins, when containing a free 5'-phosphorylated end
and a complex of four .PHI.29 proteins, will catalyze the
isothermal replication of 50-100 kb of single-stranded DNA from the
site of origin, owing to the high processivity of .PHI.29 DNA
polymerase (P2). This replicated ssDNA contains a 5'-P-linked
.PHI.29 terminal protein (P3) that can be used as a handle to
isolate the replicated ssDNA from the rest of the sample. Once
isolated, conventional NGS library or CGH array approaches can be
used to interrogate the replicated ssDNA. This approach will be
particularly useful for determining what sequences lie a
significant distance downstream of a known sequence. For example, a
small portion of a bacterial genome present in a complex
metagenomic sample may be known, but more sequence of this
bacterium is desired. Alternatively, a suspected chromosomal
rearrangement may lie downstream of a particular locus, and the
exact sequence surrounding the rearrangement may be desired.
[0115] In more detail, the origins of replication for .PHI.29 are
191 bp and 194 bp sequences derived from the left and right ends of
the .PHI.29 genome, respectively. Smaller functional regions of
these .PHI.29 replication origins have been partially mapped, and
the smallest known functional .PHI.29 replication origin is 68 nt.
These sequences bind to a complex of four .PHI.29 proteins--the
.PHI.29 DNA polymerase and terminal protein mentioned above, P5 (a
single-stranded DNA binding protein) and P6 (a double-stranded DNA
binding protein).
[0116] The .PHI.29 replication complex involves coating and
unwinding the .PHI.29 replication origin by the concerted actions
of P5 and P6, and covalent attachment of P3 to the 5' end of the
DNA, which serves to prime replication by P2 polymerase. No
oligonucleotides are necessary to prime .PHI.29 transcription due
to the presence of P3, thus .PHI.29 polymerase can extend multiple
.PHI.29 replication origin integrants in parallel using the same
four factors.
[0117] To generate a suitable set of ends for the integrated origin
of replication, a unique cut site may be engineered into the
.PHI.29 replication origin. One solution may be to incorporate a
deoxyuracil in place of a thymidine at an appropriate location in
the .PHI.29 origin. Addition of uracil deglycosylase (USER) will
remove the uracil, and the second strand can be cleaved by addition
of T7 endonuclease I. This will provide a terminus for D29
polymerase entry in conjunction with the other accessory
factors.
[0118] This cas9::transposase-.PHI.29 ORI approach could be applied
to a slightly different application by adding two .PHI.29 ORIs to
FFPE slides with reversed formaldehyde crosslinks. This system
could be applied for the isothermal amplification and visualization
(fluorescent or otherwise-detectable nucleotides) of large
chromosomal amplicons, which may be detectable in situ by
microscopic evaluation, or could otherwise provide a minimally
destructive method for interrogating precious clinical samples by
NGS.
[0119] Depending on how the method is implemented and how the
products are sequenced, the new sequence insertions may be
distributed along the product molecule with an average spacing that
is in the range of 50 bases to 20 kb, e.g., 100 bases to 10 kb or
200 bases to 2 kb, for example. In other embodiments, the new
sequence insertions may be distributed along the product molecule
with an average spacing of 10 kb to 100 kb, for example.
[0120] The Cas9-protein may be fused to Sleeping Beauty, Piggybac
or Tn5 transposons, among others. The disclosure of US20120208724
is incorporated by reference herein for all purposes.
Method for Overcoming a Transformation Barrier in a Bacterial
Host
[0121] A method for overcoming a transformation barrier in a
bacterial host is provided. In these embodiments, the method may
comprise:
[0122] (a) identifying a bacterial host that is recalcitrant to
transformation with a particular plasmid;
[0123] (b) knocking out the Cas9/CRISPR system in the bacterial
host,
[0124] (c) transforming the host with the knocked out Cas9/CRISPR
system with the plasmid.
[0125] In certain embodiments, the Cas9/CRISPR system is knocked
out by making a mutation in a Cas9 coding sequence in the genome of
the host.
[0126] In certain embodiments, the Cas9/CRISPR system may be
knocked out by expressing an anti-Cas9 protein into the host
[0127] In certain embodiments, the host is an extremophile.
[0128] In certain embodiments, the host is an acidophile, an
alkaliphile, an anaerobe, a cryptoendolith, a halophile, a
hyperthermophile, a hypolith, a lithoautotroph, a metallotolerant,
an oligotroph, an osmophile, a piezophile, a psychrophile, a
radioresistant organism, a thermophile, a thermoacidophile or a
xerophile.
[0129] In certain embodiments, the plasmid comprises an origin of
replication and an antibiotic resistance gene.
[0130] Bioinformatic analyses suggest CRISPR-based innate immunity
systems exist in >50% of all bacterial species and >90% of
archaebacteria. In some species CRISPR represents a barrier to
transformation, and that by eliminating CRISPR-based innate
immunity we can make some microbial species amenable to genetic
manipulation. A recent report described various anti-CRISPR
proteins derived from phage (Bondy-Denomy et al, 2013 Nature
493:429-432). We propose that expression or co-transformation of
anti-CRISPR protein or DNA can be used to overcome the
transformation barrier in certain hosts. Using anti-CRISPR proteins
to overcome barriers to transformation has a number of advantages:
First, the presence of a CRISPR system in the host organism can be
easily determined by genomic DNA sequence analysis. Second,
co-expression of anti-CRISPR requires no a priori manipulation of
the host genome. Third, inducible expression of anti-CRISPR
proteins allows for negative selection of plasmids (ie plasmids are
destroyed in a CRISPR-dependent fashion when anti-CRISPR protein
expression is shut off).
[0131] In one embodiment, a plasmid encoding one or more
anti-CRISPR proteins along with an antibiotic resistance gene and
gene(s) of interest are transformed into a new host and
transformants are selected based on antibiotic resistance. The
presence of antibiotic resistant clones and the loss of antibiotic
resistance when anti-CRISPR gene expression is turned off are
phenotypes that indicate the barrier to transformation has been
effectively eliminated. In a second embodiment, anti-CRISPR protein
is co-transformed with said plasmid in order to ensure the plasmid
is not restricted by an active CRISPR system before gene expression
is established.
[0132] The method described above employed to make any bacterium
competent for transformation with, e.g., a nucleic acid such as a
vector that has an appropriate origin of replication. See, e.g.,
Johnsborg et al. Res. Microbiol 2007 158: 767-78, which is
incorporated by reference. In some embodiments, the method finds
particular use in transforming extremophilic bacteria or
archaebacteria such as an acidophile (an organism with optimal
growth at pH levels of 3 or below), an alkaliphile (an organism
with optimal growth at pH levels of 9 or above), an anaerobe (an
organism that does not require oxygen for growth such as
Spinoloricus Cinzia) a cryptoendolith (an organism that lives in
microscopic spaces within rocks), a halophile (an organism
requiring at least 0.2M concentrations of salt (NaCl) for growth),
hyperthermophile (an organism that can thrive at temperatures
between 80-122.degree. C.), a hypolith (an organism that lives
underneath rocks in cold deserts), a lithoautotroph (an organism
whose sole source of carbon is carbon dioxide and exergonic
inorganic oxidation), a metallotolerant (which is capable of
tolerating high levels of dissolved heavy metals in solution), an
oligotroph (an organism capable of growth in nutritionally limited
environments), an osmophile (an organism capable of growth in
environments with a high sugar concentration), a piezophile (an
organism that lives optimally at high pressures such as those deep
in the ocean or underground), a psychrophile (an organism capable
of survival, growth or reproduction at temperatures of -15.degree.
C. or lower for extended periods), a radioresistant organism that
is resistant to high levels of ionizing radiation, a thermophile
(an organism that can thrive at temperatures between 45-122.degree.
C.), a thermoacidophile (a combination of thermophile and
acidophile that prefer temperatures of 70-80.degree. C. and pH
between 2 and 3) and a xerophile (which grows in extremely dry,
desiccating conditions).
Method for SNP Detection
[0133] Also provided herein is a method for SNP detection.
[0134] In some embodiments, this method comprises:
[0135] (a) contacting a genomic sample that comprises a polymorphic
site with a Cas9-gRNA complex that comprises an allele-specific
guide RNA; and
[0136] (b) determining whether the Cas9-gRNA complex cleaves the
polymorphic site, wherein cleavage of the polymorphic site
indicates the allele of the SNP at the polymorphic site.
[0137] In some embodiments, the genomic sample comprises mammalian
genomic DNA.
[0138] In some embodiments, the genomic sample comprises human
genomic DNA.
[0139] In some embodiments, the genomic sample comprises plant
genomic DNA.
[0140] In some embodiments, the SNP is associated with a
disease.
[0141] In some embodiments, the method comprises contacting the
genomic sample with a plurality of Cas9- guide RNA complexes,
wherein each guide RNA targets a particular SNP allele.
[0142] In some embodiments, the method comprises contacting the
genomic sample with at least 1,000 Cas9- guide RNA complexes, each
targeting a different SNP.
[0143] In some embodiments, the method cleavage products are
analyzed by gel electrophoresis, mass spectrometry or by
sequencing.
[0144] In this method, the term "single nucleotide polymorphism,"
or "SNP" or "SNP site" for short, refers to the single nucleotide
position in a genomic sequence for which two or more alternative
alleles are present at appreciable frequency (e.g., at least 1%) in
a population. An "SNP allele" refers to the identity of the
nucleotide of SNP. A "first allele" and a "second allele" of a SNP
are different alleles, i.e., they have different SNP nucleotides.
When a Cas9-gRNA complex cleaves near a SNP "only if a first allele
is present," the Cas9-gRNA complex cleaves at a first allele of the
SNP and not at a different (i.e., second) allele of the SNP.
[0145] Since the nucleotide sequences of hundreds of thousands of
SNPs from humans, other mammals (e.g., mice), and a variety of
different plants (e.g., corn, rice and soybean), are known (see,
e.g., Riva et al 2004, A SNP-centric database for the investigation
of the human genome BMC Bioinformatics 5:33; McCarthy et al. 2000
The use of single-nucleotide polymorphism maps in pharmacogenomics
Nat Biotechnology 18:505-8) and are available in public databases
(e.g., NCBI's online site-specific nicking endonuclease dbSNP
database, and the online database of the International HapMap
Project; see also Teufel et al. 2006 Current bioinformatics tools
in genomic biomedical research Int. J. Mol. Med. 17:967-73), and
several SNPs lie proximal to a PAM motif, designing guide RNAs that
recognize and cleave at particular alleles of a SNP would be well
within the skill of one skilled in the art.
[0146] Disease-associated allele variants can sometimes be
identified as SNPs that change the restriction pattern of said
sequence when digested with a given restriction enzyme and can be
detected by a technique called Restriction Fragment Length
Polymorphism (RFLP) mapping. Other allele variants of the same gene
may generate SNPs cleavable by the same restriction enzyme, or a
different restriction enzyme, or with no available enzymes.
Consequently, there is no way to do a single RFLP digest to perform
comprehensive SNP analysis of all known disease-associated allele
variants. SNP analysis is also commonly used to classify species
from environmental samples.
[0147] SNP analysis could be performed using Cas9 programmed with a
library of guide RNAs that can uniquely identify known SNPs. Use of
Cas9 allows for universal reaction conditions and ease of
automation. Additionally, because Cas9 likely uses a scanning
method for homology search, overlapping guide mRNAs should not
interfere extensively with each other as they would with standard
hybridization methods.
[0148] For a given SNP, a guide mRNA can be designed such that the
unique sequence represented by the SNP is incorporated into the
guide. Such a guide mRNA should program a Cas9 complex to cleave
the SNP sequence but not the reference sequence. Digestions
targeting multiple SNPs could be done in parallel (multiplexed) so
that the presence/absence of a panel of SNP variants could be
determined in a single reaction. Analysis of digestion products
could be performed by gel electrophoresis, mass spectrometry, or
other methods.
[0149] In certain embodiments, the method may be done using a panel
of guide RNAs, where each guide RNA targets a particular SNP
allele. The method may be used to analyze at least 2, at least 10,
at least 1,000, at least 10,000 or at least 100,000 or more
different SNPs in a sample, in parallel. The resultant fragments
can be analyzed using any suitable method, including gel
electrophoresis, mass spectrometry or sequencing.
Method for detecting a microbe
[0150] Also provided herein is a method for detecting a microbe in
a sample.
[0151] Some embodiments of the method comprise:
[0152] (a) contacting a sample comprising microbial DNA with a
Cas9-gRNA complex and a microbe-specific guide RNA; and
[0153] (b) determining whether the Cas9-gRNA complex cleaves the
microbial DNA, wherein cleavage of the microbial DNA indicates the
microbe is in the sample.
[0154] In some embodiments, the microbial DNA is of unknown
species.
[0155] In some embodiments, the microbe-specific guide RNA
specifically hybridizes to a microbe that is associated with a
disease.
[0156] In some embodiments, the method comprises contacting the
sample sample with a plurality of Cas9- microbe-specific guide RNA
complexes, wherein each guide RNA targets a different microbe.
[0157] In some embodiments, the method comprises contacting the
sample with at least 1,000 Cas9- microbe-specific guide RNA
complexes, each targeting a different microbe.
[0158] In some embodiments, the method cleavage products are
analyzed by gel electrophoresis, mass spectrometry or by
sequencing.
[0159] In this embodiment, the term "microbe," as used herein,
refers to a microorganism. The term includes bacteria, fungi,
archaea, and protists. The term "microbe" includes pathogenic
bacteria, causing diseases such as plague, tuberculosis and
anthrax; protozoa, causing diseases such as malaria, sleeping
sickness and toxoplasmosis; and also fungi causing diseases such as
ringworm, candidiasis or histoplasmosis, for example.
[0160] The guide RNAs used are microbe-specific in that they are
capable of distinguishing between different microbes, where the
term "different microbes" refers to microbes that are distinct from
each other because they belong to a different genus, or to a
different species or to a different strain. Two microbes that
belong to different genera are considered to be different, microbes
that belong to the same genus but to different strains are
considered to be different, microbes that belong to the same genus
and species but to different strains are also considered to be
different.
[0161] Many pathogens can cause food-borne illness and can be
identified by specific PCR-based assays that identify unique DNA
sequences in said pathogens' genome (see for example, Naravaneni
and Jamil, J Med. Microbiol. 2005). In general, pathogens represent
an extremely small fraction of the total sample but can still be
dangerous at these low levels. Most technologies aiming to identify
pathogens based on genomic sequences have difficulty in both
precision and sensitivity. Additionally, rapid evolution of
pathogens leads to rapid obsolescence of detection assays. Cas9 can
be programmed with a library of guide mRNAs that can uniquely
identify a large number of pathogens can provide a single, rapid
assay for identification of said pathogens.
[0162] Cas9/crRNA can be used as an efficient targeting system to
place desired protein and nucleic acid payloads (fused to Cas9) in
specific locations on target DNAs. Here we use Cas9/crRNA as a tool
to modify and subsequently identify rare DNA sequences in complex
mixtures.
[0163] A Cas9-based assay addresses the precision problem by
allowing one to design a large number of target sites in any
pathogen genome to generate fragments for sequence analysis. There
is no limitation for selecting unique sequences with an appropriate
melting temperature that has proved difficult for any
hybridization-based technologies. Likewise, because each target
fragment must contain the precise .about.24 base pair Cas9
recognition sequence at each end of the target, the potential for
off-target cleavage and generation of fragments amenable to
sequencing is rare. Sensitivity in a Cas9-based assay should be far
superior to raw sequencing, since the vast majority of DNA in a
sample will not be amenable for sequencing and because
amplification of the small fraction of Cas9-generated fragments
prior to sequencing is available.
[0164] In one embodiment, DNA is extracted from a food sample or
culture and sheared into small fragments and treated with a
chemical reagent that specifically reacts with 3' OH groups at the
DNA ends and inhibits ligation (for example, 4-nitro isatoic
anhydride, as described in Invention disclosure # 20130128).
Sheared fragments are then digested with a pool of Cas9 programmed
with a guide mRNA library specific to from one to 100 unique
pathogens. For each pathogen, pairs of sequences have been selected
so that each pair is unique to a single pathogen of interest or to
a family of pathogens, and is separated by 40 to 250 nucleotides.
For each pathogen or pathogen family, from one to 1000 target pairs
may be selected. Digested fragments are ligated at high efficiency
to common PCR/sequencing primers, whereas DNA fragments with
derivitized ends are not. The sample is then subject to PCR using
said common primers to generate a binary output (PCR
positive=pathogen positive) or subjected to sequencing for precise
identification of one or more pathogen species.
[0165] In certain embodiments, the method may be done using a panel
of guide RNAs, where each guide RNA targets a particular microbe.
The method may be used to analyze at least 2, at least 10, at least
1,000, at least 10,000 or at least 100,000 or more different
microbes in a sample, in parallel. The resultant fragments can be
analyzed using any suitable method, including gel electrophoresis,
mass spectrometry or sequencing.
[0166] Guide RNAs used in this subject method may be designed by
utilizing the genome sequence information as well as expressed gene
sequence information available at several public and private
databases, for example. For example, genomic sequence information
is available via the Microbe Genome Sequencing Project, Department
of Energy, U.S.A. and from NCBI. Expressed gene sequence
information is available at GenBank. Additionally, expressed gene
sequences can be derived from gene expression profiling of microbes
of interest. Microarrays representing the genome of a variety of
microbes as well as custom microarrays for microbes of interest are
available from numerous vendors.
[0167] The above described method is useful for the analysis of
samples in a variety of diagnostic, drug discovery, and research
applications. The above described method is useful for the analysis
of biological samples. The term "biological sample," as used
herein, refers to a sample obtained from an organism or from
components (e.g., cells) of an organism. The sample may be of any
biological tissue or fluid. In some cases, the sample will be a
"clinical sample" which is a sample derived from a patient. Such
samples include, but are not limited to, sputum, blood, blood cells
(e.g., white blood cells), tissue or fine needle biopsy samples,
urine, peritoneal fluid, and pleural fluid, or cells there from.
Biological samples may also include sections of tissues such as
frozen sections taken for histological purposes. The subject method
also finds use in determining the identity of microbes in water,
sewage, air samples, food products, including animals, vegetables,
seeds, etc., soil samples, plant samples, microbial culture
samples, cell culture samples, tissue culture samples, as well as
in human medicine, veterinary medicine, agriculture, food science,
bioterrorism, and industrial microbiology, etc. The subject method
allows identification of hard to culture microbes since culturing
the microbes is not necessary. Consequently, the subject method
provides for a rapid detection of microbes in a sample with no
waiting period for culturing microbes.
[0168] Microbes that might be identified using the subject methods,
compositions and kits include but are not limited to: a plurality
of species of Gram (+) bacteria, plurality of species of Gram (-)
bacteria, a plurality of species of bacteria in the family
Enterobacteriaceae, a plurality of species of bacteria in the genus
Enterococcus, a plurality of species of bacteria in the genus
Staphylococcus, and a plurality of species of bacteria in the genus
Campylobacter, Escherichia coli (E. coli), E. coli of various
strains such as, K12-MG1655, CFT073,O157:H7 EDL933, 0157:H7
VT2-Sakai, etc., Streptococcus pneumoniae, Pseudomonas aeruginosa,
Staphylococcus aureus, coagulase-negative staphylococci, a
plurality of Candida species including C. albicans, C. tropicalis,
C. dubliniensis, C. viswanathii, C. parapsilosis, Klebsiella
pneumoniae, a plurality of Mycobacterium species such as M.
tuberculosis, M. bovis, M. bovis BCG, M. scrofulaceum, M. kansasii,
M. chelonae, M. gordonae, M. ulcerans, M. genavense, M. xenoi, M.
simiae, M. fortuitum, M. malmoense, M. celatum, M. haemophilum and
M. africanum, Listeria species, Chlamydia species, Mycoplasma
species, Salmonella species, Brucella species, Yersinia species,
etc. Thus, the subject method enables identification of microbes to
the level of the genus, species, sub-species, strain or variant of
the microbe.
Method of Screening for a Cas9 Variant
[0169] Also provided herein is a method of screening for a Cas9
variant that has improved activity (e.g., one that produces more
blunt ended breaks).
[0170] In certain embodiments, this method comprises:
[0171] (a) combining, in a cell, a test Cas9 protein and a Cas9
guide RNA with a target plasmid that comprises a marker that is
disrupted by an insert, wherein: [0172] (i) the marker can be
reconstituted by blunt end cleavage at sites that flank the insert
and re-ligation of the marker sequence, and [0173] (ii) the Cas9
guide RNA targets the Cas9 protein to the sites that flank the
insert; and
[0174] (b) determining whether the cell expresses the marker.
[0175] In some embodiments, the marker is an antibiotic marker.
[0176] In some embodiments, the marker is a colorigenic marker.
[0177] In some embodiments, the marker is a light-emitting
marker.
[0178] In some embodiments, the test Cas9 protein is variant of
wild type Cas9 protein that contains up to 20 amino acid
substitutions relative to the wild type Cas9 protein.
[0179] In some embodiments, the method comprises:
[0180] (a) combining, in a population of cells, a library of
nucleic acids that encode at least 100 Cas9 variants and a Cas9
guide RNA with a target plasmid that comprises a disrupted marker,
wherein (i) the marker is disrupted by an insert, (ii) the marker
can be reconstituted by blunt end cleavage at sites that flank the
insert and re-ligation of the marker sequence, and (iii) the Cas9
guide RNA targets the Cas9 protein to the sites that flank the
insert; and
[0181] (b) screening the cells for expression of the marker
[0182] In some embodiments, the method may further comprise
sequencing cells expressing the marker, thereby providing the amino
acid sequence of an improved Cas9 protein.
[0183] Cleavage by the Cas9-gRNA complex comprises several steps:
tracRNA loading; DNA binding; specific target selection; cleavage
by the RuvC-like domain; cleavage by the HNH domain; and release of
the cleaved DNA. Inefficiencies in any of these steps may lead to
reduced cleavage by Cas9, and some of the steps, such as tracRNA
loading, are specific to Cas9.
[0184] Cas9 can be evolved in vivo. First, a suitable host such as
E. coli can be transformed with a library of protein expression
vectors that each contain a variant of Cas9 as well as a
corresponding tracRNA. The Cas9 variants can be made by a directed
evolution method, e.g., a method described in Often et al
(Biomolecular Engineering 2005 22: 1-9), Reetz et al
[0185] (Nature Prot. 2007 2: 891-903), Stemmer (Nature 1994 370:
389-391) and Labrou (Curr. Protein Pept. Sci. 2010 11: 91-100). The
method may involve error-prone PCR or DNA shuffling, for example
and in particular cases may be adapted from phage display, enzyme
engineering or zinc finger technologies. Many molecular techniques
may be employed in this method, e.g., random PCR mutagenesis, see,
e.g., Rice et al. (1992) Proc. Natl. Acad. Sci. USA 89:5467-5471;
or, combinatorial multiple cassette mutagenesis, see, e.g., Crameri
et al. (1995) Biotechniques 18:194-196. Alternatively, nucleic
acids, e.g., genes, can be reassembled after random, or
"stochastic," fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242;
6,287,862; 6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238;
5,605,793. In alternative aspects, modifications, additions or
deletions are introduced by error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly (e.g., GeneReassembly,
see, e.g., U.S. Pat. No. 6,537,776), gene site saturation
mutagenesis (GSSM), synthetic ligation reassembly (SLR),
recombination, recursive sequence recombination,
phosphothioate-modified DNA mutagenesis, uracil-containing template
mutagenesis, gapped duplex mutagenesis, point mismatch repair
mutagenesis, repair-deficient host strain mutagenesis, chemical
mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification
mutagenesis, artificial gene synthesis, ensemble mutagenesis,
chimeric nucleic acid multimer creation, and/or a combination of
these and other methods.
[0186] Ideally the cleavage site of Cas9 (as determined by the
tracRNA) will be chosen such that the host genome has few or no
copies that would be targeted by the enzyme. However, the cells are
also transformed with a modified gene for antibiotic resistance,
such as the beta-lactamase gene which confers resistance to
ampicillin. This resistance gene may reside on the same expression
vector as the Cas9, or on another vector, or in the genome. The
modification of the resistance gene comprises an interrupting
sequence flanked by Cas9 recognition sites. In the absence of Cas9
activity, the interruption in the resistance gene will preclude
production of an active resistance protein. However, in the
presence of Cas9 activity, the interrupting sequence can be excised
by cleavage of both flanking sites and subsequent ligation of the
antibiotic resistance gene. In order for the cells to produce
active antibiotic resistance genes, the Cas9 protein must
efficiently complete the cleavage steps outlined above, leaving DNA
ends that are suitable substrates for DNA ligase. Cas9 variants
that leave uneven DNA ends, or variants that do not release the
cleaved DNA will not leave DNA ends suitable for ligation. In order
to select for more specific cleavage by Cas9, single-base variants
of the Cas9 target sequence could be included in the Cas9 and/or
antibiotic resistance vector. If the Cas9 variant in that cell
induces cleavage of these off-target sequences, expression of the
Cas9 protein or antibiotic resistance protein will be reduced.
[0187] This description is an outline of the method, and the
skilled artisan will understand that the expression host, vectors,
or resistance genes could be varied without substantially altering
the method. Furthermore, in embodiments it may be preferable to use
a different reporter gene such as beta-galactosidase or green
fluorescent protein in place of the antibiotic resistance gene. In
those cases, a colorimetric or fluorescence assay could be used to
assay Cas9 activity in vivo.
[0188] This method may in certain embodiments comprise: combining,
in a population of cells, a library of nucleic acids that encode
Cas9 variants (e.g., at least 2, at least 10, at least 1,000, at
least 10,000 or at least 100,000 or more variants) and a Cas9 guide
RNA with a target plasmid that comprises a disrupted marker,
wherein the marker is disrupted by an insert, wherein the marker
can be reconstituted by blunt end cleavage at sites that flank the
insert and re-ligation of the marker sequence, and the Cas9 guide
RNA targets the Cas9 protein to the sites that flank the insert.
After the cells have been made, the cells can be selected for
expression of the marker (which would be made by cleavage by Cas9,
followed by repair). The Cas9-encoding nucleic acid of the selected
cells can then be sequenced, thereby providing the amino acid
sequence of an improved Cas9 protein.
* * * * *