U.S. patent application number 13/006625 was filed with the patent office on 2011-08-18 for recognition sequences for i-crei-derived meganucleases and uses thereof.
Invention is credited to Derek JANTZ, James J. SMITH.
Application Number | 20110202479 13/006625 |
Document ID | / |
Family ID | 41550694 |
Filed Date | 2011-08-18 |
United States Patent
Application |
20110202479 |
Kind Code |
A1 |
JANTZ; Derek ; et
al. |
August 18, 2011 |
RECOGNITION SEQUENCES FOR I-CREI-DERIVED MEGANUCLEASES AND USES
THEREOF
Abstract
Methods of cleaving double-stranded DNA that can be recognized
and cleaved by a rationally-designed, I-CreI-derived meganuclease
are provided. Also provided are recombinant nucleic acids, cells,
and organisms containing such recombinant nucleic acids, as well as
cells and organisms produced using such meganucleases. Also
provided are methods of conducting a custom-designed,
I-CreI-derived meganuclease business.
Inventors: |
JANTZ; Derek; (Durham,
NC) ; SMITH; James J.; (Durham, NC) |
Family ID: |
41550694 |
Appl. No.: |
13/006625 |
Filed: |
January 14, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US2009/050566 |
Jul 14, 2009 |
|
|
|
13006625 |
|
|
|
|
61080453 |
Jul 14, 2008 |
|
|
|
Current U.S.
Class: |
705/500 ;
435/252.3; 435/254.11; 435/325; 435/366; 435/419; 435/91.1;
435/91.53 |
Current CPC
Class: |
C12Q 1/6811 20130101;
G06Q 99/00 20130101; C12N 9/22 20130101 |
Class at
Publication: |
705/500 ;
435/91.53; 435/366; 435/325; 435/419; 435/254.11; 435/91.1;
435/252.3 |
International
Class: |
C12P 19/34 20060101
C12P019/34; C12N 5/10 20060101 C12N005/10; C12N 1/15 20060101
C12N001/15; C12N 1/21 20060101 C12N001/21; G06Q 90/00 20060101
G06Q090/00 |
Claims
1. A method for cleaving a double-stranded DNA comprising: (a)
identifying in said DNA at least one recognition site for a
rationally-designed I-CreI-derived meganuclease with altered
specificity relative to I-CreI, wherein said recognition site is
not cleaved by a naturally-occurring I-CreI, wherein said
recognition site has a four base pair central sequence selected
from the group consisting of TTGT, TTAT, TCTT, TCGT, TCAT, GTTT,
GTCT, GGAT, GAGT, GAAT, ATGT, TTTC, TTCC, TGAC, TAAC, GTTC, ATAT,
TCGA, TTAA, GGGC, ACGC, CCGC, CTGC, ACAA, ATAA, AAGA, ACGA, ATGA,
AAAC, AGAC, ATCC, ACTC, ATTC, ACAT, GAAA, GGAA, GTCA, GTTA, GAAC,
ATAT, TCGA, TTAA, GCCC, GCGT, GCGG and GCAG; (b) providing said
rationally-designed meganuclease; and (c) contacting said DNA with
said rationally-designed meganuclease; whereby said
rationally-designed meganuclease cleaves said DNA.
2. (canceled)
3. The method of claim 1, wherein said DNA cleavage is in
vitro.
4. The method of claim 1, wherein said DNA is selected from the
group consisting of a PCR product; an artificial chromosome;
genomic DNA isolated from bacteria, fungi, plants, or animal cells;
and viral DNA.
5. The method of claim 1, wherein said DNA cleavage is in vivo.
6. The method of claim 5, wherein said DNA is present in a cell
selected from the group consisting of a bacterial, fungal, plant
and animal cell.
7. The method of claim 5, wherein said DNA is present in a nucleic
acid selected from the group consisting of a plasmid, a prophage
and a chromosome.
8. The method of claim 1, wherein said four base pair DNA sequence
is selected from the group consisting of GTGT, GTAT, TTAG, GTAG,
TTAC, TCTC, TCAC, GTCC, GTAC, TCGC, AAGC, GAGC, GCGC, GTGC, TAGC,
TTGC, ATGC, ACAC, ATAC, CTAA, CTAC, GTAA, GAGA, GTGA, GGAC, GTAC,
GCGA, GCTT, GCTC, GCGC, GCAC, GCTA, GCAA and GCAT.
9. The method of claim 1, further comprising rationally-designing
said I-CreI-derived meganuclease to recognize said recognition
site.
10. The method of claim 1, further comprising producing said
rationally-designed I-CreI-derived meganuclease.
11. A cell transformed with a nucleic acid comprising, in order: a)
a first 9 base pair DNA sequence which can be bound by an
I-CreI-derived meganuclease monomer or by a first domain from a
single-chain I-CreI-derived meganuclease; b) a four base pair DNA
sequence selected from the group consisting of GTGT, GTAT, TTAG,
GTAG, TTAC, TCTC, TCAC, GTCC, GTAC, TCGC, AAGC, GAGC, GCGC, GTGC,
TAGC, TTGC, ATGC, ACAC, ATAC, CTAA, CTAC, GTAA, GAGA, GTGA, GGAC,
GTAC, GCGA, GCTT, GCTC, GCGC, GCAC, GCTA, GCAA and GCAT; and c) a
second 9 base pair DNA sequence which can be bound by an
I-CreI-derived meganuclease monomer or by a second domain from the
single-chain I-CreI-derived meganuclease, wherein the second 9 base
pair DNA sequence is in the reverse orientation relative to the
first.
12. A cell containing an exogenous nucleic acid sequence integrated
into its genome, comprising, in order: a) a first exogenous 9 base
pair DNA sequence which can be bound by an I-CreI-derived
meganuclease monomer or by a first domain from a single-chain
I-CreI-derived meganuclease; b) an exogenous four base pair DNA
sequence selected from the group consisting of GTGT, GTAT, TTAG,
GTAG, TTAC, TCTC, TCAC, GTCC, GTAC, TCGC, AAGC, GAGC, GCGC, GTGC,
TAGC, TTGC, ATGC, ACAC, ATAC, CTAA, CTAC, GTAA, GAGA, GTGA, GGAC,
GTAC, GCGA, GCTT, GCTC, GCGC, GCAC, GCTA, GCAA and GCAT; and a) a
second exogenous 9 base pair DNA sequence which can be bound by an
I-CreI-derived meganuclease monomer or by a second domain from the
single-chain I-CreI-derived meganuclease, wherein the second 9 base
pair DNA sequence is in the reverse orientation relative to the
first.
13. The cell of claim 11, wherein said nucleic acid is a
plasmid.
14. The cell of claim 11, wherein said nucleic acid is an
artificial chromosome.
15. The cell of claim 11, wherein said nucleic acid is integrated
into the genomic DNA of said cell.
16. The cell of claim 11, wherein said nucleic acid is a viral
nucleic acid.
17. The cell of claim 11, wherein said cell is selected from the
group consisting of a human cell, a non-human animal cell, a plant
cell, a bacterial cell, and a fungal cell.
18. The cell of claim 11, wherein said four base pair DNA sequence
is selected from the group consisting of TTGT, TTAT, TCTT, TCGT,
TCAT, GTTT, GTCT, GGAT, GAGT, GAAT, ATGT, TTTC, TTCC, TGAC, TAAC,
GTTC, ATAT, TCGA, TTAA, GGGC, ACGC, CCGC, CTGC, ACAA, ATAA, AAGA,
ACGA, ATGA, AAAC, AGAC, ATCC, ACTC, ATTC, ACAT, GAAA, GGAA, GTCA,
GTTA, GAAC, ATAT, TCGA, TTAA, GCCC, GCGT, GCGG and GCAG.
19. The cell of claim 18, wherein said four base pair DNA sequence
is selected from the group consisting of GTGT, GTAT, TTAG, GTAG,
TTAC, TCTC, TCAC, GTCC, GTAC, TCGC, AAGC, GAGC, GCGC, GTGC, TAGC,
TTGC, ATGC, ACAC, ATAC, CTAA, CTAC, GTAA, GAGA, GTGA, GGAC, GTAC,
GCGA, GCTT, GCTC, GCGC, GCAC, GCTA, GCAA and GCAT.
20. A method of conducting a custom-designed, I-CreI-derived
meganuclease business comprising: (a) receiving a DNA sequence into
which a double-strand break is to be introduced by a
rationally-designed I-CreI-derived meganuclease; (b) identifying in
said DNA sequence at least one recognition site for a
rationally-designed I-CreI-derived meganuclease with altered
specificity relative to I-CreI, wherein said recognition site is
not cleaved by a naturally-occurring I-CreI, wherein said
recognition site has a four base pair central sequence selected
from the group consisting of TTGT, TTAT, TCTT, TCGT, TCAT, GTTT,
GTCT, GGAT, GAGT, GAAT, ATGT, TTTC, TTCC, TGAC, TAAC, GTTC, ATAT,
TCGA, TTAA, GGGC, ACGC, CCGC, CTGC, ACAA, ATAA, AAGA, ACGA, ATGA,
AAAC, AGAC, ATCC, ACTC, ATTC, ACAT, GAAA, GGAA, GTCA, GTTA, GAAC,
ATAT, TCGA, TTAA, GCCC, GCGT, GCGG and GCAG; and (c) providing said
rationally-designed meganuclease.
21. The method of claim 20, further comprising rationally-designing
said I-CreI-derived meganuclease to recognize said recognition
site.
22. The method of claim 20, further comprising producing said
rationally-designed meganuclease.
23. The method of claim 20, wherein the rationally-designed
meganuclease is provided to the same party from which said DNA
sequence has been received.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of International
Application PCT/US2009/50566 filed on Jul. 14, 2009, which claims
the benefit of U.S. Provisional Application No. 61/080,453, filed
Jul. 14, 2008, the entire disclosures of which are incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to the field of molecular biology and
recombinant nucleic acid technology. In particular, the invention
relates to DNA sequences that can be recognized and cleaved by a
non-naturally-occurring, rationally-designed, I-CreI-derived homing
endonuclease and methods of using same. The invention also relates
to methods of producing recombinant nucleic acids, cells, and
organisms using such meganucleases which cleave such DNA sites. The
invention further relates to methods of conducting a
custom-designed, I-CreI-derived meganuclease business.
BACKGROUND OF THE INVENTION
[0003] Genome engineering requires the ability to insert, delete,
substitute and otherwise manipulate specific genetic sequences
within a genome, and has numerous therapeutic and biotechnological
applications. The development of effective means for genome
modification remains a major goal in gene therapy, agrotechnology,
and synthetic biology (Porteus et al. (2005), Nat. Biotechnol. 23:
967-73; Tzfira et al. (2005), Trends Biotechnol. 23: 567-9;
McDaniel et al. (2005), Curr. Opin. Biotechnol. 16: 476-83). A
common method for inserting or modifying a DNA sequence involves
introducing a transgenic DNA sequence flanked by sequences
homologous to the genomic target and selecting or screening for a
successful homologous recombination event. Recombination with the
transgenic DNA occurs rarely, but can be stimulated by a
double-stranded break in the genomic DNA at the target site.
Numerous methods have been employed to create DNA double-stranded
breaks, including irradiation and chemical treatments. Although
these methods efficiently stimulate recombination, the
double-stranded breaks are randomly dispersed in the genome, which
can be highly mutagenic and toxic. At present, the inability to
target gene modifications to unique sites within a chromosomal
background is a major impediment to successful genome
engineering.
[0004] One approach to achieving this goal is stimulating
homologous recombination at a double-stranded break in a target
locus using a nuclease with specificity for a sequence that is
sufficiently large to be present at only a single site within the
genome (see, e.g., Porteus et al. (2005), Nat. Biotechnol. 23:
967-73). The effectiveness of this strategy has been demonstrated
in a variety of organisms using chimeric fusions between an
engineered zinc finger DNA-binding domain and the non-specific
nuclease domain of the FokI restriction enzyme (Porteus (2006), Mol
Ther 13: 438-46; Wright et al. (2005), Plant J. 44: 693-705; Urnov
et al. (2005), Nature 435: 646-51). Although these artificial zinc
finger nucleases stimulate site-specific recombination, they retain
residual non-specific cleavage activity resulting from
under-regulation of the nuclease domain and frequently cleave at
unintended sites (Smith et al. (2000), Nucleic Acids Res. 28:
3361-9). Such unintended cleavage can cause mutations and toxicity
in the treated organism (Porteus et al. (2005), Nat. Biotechnol.
23: 967-73).
[0005] A group of naturally-occurring nucleases which recognize
15-40 base-pair cleavage sites commonly found in the genomes of
plants and fungi may provide a less toxic genome engineering
alternative. Such "meganucleases" or "homing endonucleases" are
frequently associated with parasitic DNA elements, such as group 1
self-splicing introns and inteins. They naturally promote
homologous recombination or gene insertion at specific locations in
the host genome by producing a double-stranded break in the
chromosome, which recruits the cellular DNA-repair machinery
(Stoddard (2006), Q. Rev. Biophys. 38: 49-95). Meganucleases are
commonly grouped into four families: the LAGLIDADG family, the
GIY-YIG family, the His-Cys box family and the HNH family. These
families are characterized by structural motifs, which affect
catalytic activity and recognition sequence. For instance, members
of the LAGLIDADG family are characterized by having either one or
two copies of the conserved LAGLIDADG motif (see Chevalier et al.
(2001), Nucleic Acids Res. 29(18): 3757-3774). The LAGLIDADG
meganucleases with a single copy of the LAGLIDADG motif form
homodimers, whereas members with two copies of the LAGLIDADG motif
are found as monomers.
[0006] Natural meganucleases, primarily from the LAGLIDADG family,
have been used to effectively promote site-specific genome
modification in plants, yeast, Drosophila, mammalian cells and
mice, but this approach has been limited to the modification of
either homologous genes that conserve the meganuclease recognition
sequence (Monnat et al. (1999), Biochem. Biophys. Res. Commun. 255:
88-93) or to pre-engineered genomes into which a recognition
sequence has been introduced (Rouet et al. (1994), Mol. Cell. Biol.
14: 8096-106; Chilton et al. (2003), Plant Physiol. 133: 956-65;
Puchta et al. (1996), Proc. Natl. Acad. Sci. USA 93: 5055-60; Rong
et al. (2002), Genes Dev. 16: 1568-81; Gouble et al. (2006), J.
Gene Med. 8(5):616-622).
[0007] Systematic implementation of nuclease-stimulated gene
modification requires the use of engineered enzymes with customized
specificities to target DNA breaks to existing sites in a genome
and, therefore, there has been great interest in adapting
meganucleases to promote gene modifications at medically or
biotechnologically relevant sites (Porteus et al. (2005), Nat.
Biotechnol. 23: 967-73; Sussman et al. (2004), J. Mol. Biol. 342:
31-41; Epinat et al. (2003), Nucleic Acids Res. 31: 2952-62).
[0008] I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG family
which recognizes and cleaves a 22 base pair recognition sequence in
the chloroplast chromosome, and which presents an attractive target
for meganuclease redesign. Genetic selection techniques have been
used to modify the wild-type I-CreI recognition site preference
(Sussman et al. (2004), J. Mol. Biol. 342: 31-41; Chames et al.
(2005), Nucleic Acids Res. 33: e178; Seligman et al. (2002),
Nucleic Acids Res. 30: 3870-9, Arnould et al. (2006), J. Mol. Biol.
355: 443-58). More recently, a method of rationally-designing
mono-LAGLIDADG meganucleases was described which is capable of
comprehensively redesigning I-CreI and other such meganucleases to
target widely-divergent DNA sites, including sites in mammalian,
yeast, plant, bacterial, and viral genomes (WO 2007/047859).
[0009] The DNA sequences recognized by I-CreI are 22 base pairs in
length. One example of a naturally-occurring I-CreI recognition
site is provided in SEQ ID NO: 2 and SEQ ID NO: 3, but the enzyme
will bind to a variety of related sequences with varying affinity.
The enzyme binds DNA as a homodimer in which each monomer makes
direct contacts with a nine base pair "half-site" and the two
half-sites are separated by four base pairs that are not directly
contacted by the enzyme (FIG. 1a). Like all LAGLIDADG family
meganucleases, I-CreI produces a staggered double-strand break at
the center of its recognition sequences which results in the
production of a four base pair 3'-overhang (FIG. 1a). The present
invention concerns the central four base pairs in the I-CreI
recognition sequences (i.e. the four base pairs that become the 3'
overhang following I-CreI cleavage, or "center sequence", FIG. 1b).
In the case of the native I-CreI recognition sequence in the
Chlamydomonas reinhardtii 23S rRNA gene, this four base pair
sequence is 5'-GTGA-3'. In the interest of producing
genetically-engineered meganucleases which recognize DNA sequences
that deviate from the wild-type I-CreI recognition sequences, it is
desirable to know the extent to which the four base pair center
sequence can deviate from the wild-type sequences. A number of
published studies concerning I-CreI or its derivatives evaluated
the enzyme, either wild-type or genetically-engineered, using DNA
substrates that employed either the native 5'-GTGA-3' central
sequence or the palindromic sequence 5'-GTAC-3'. Recently, Arnould
et. al. (Arnould et al. (2007), J. Mol. Biol. 371: 49-65) reported
that a set of genetically-engineered meganucleases derived from
I-CreI cleaved DNA substrates with varying efficiencies depending
on whether the substrate sequences were centered around 5'-GTAC-3',
5'-TTGA-3', 5'-GAAA-3', or 5'-ACAC-3' (cleavage efficiency:
GTAC>ACAC>>TTGA.apprxeq.GAAA).
SUMMARY OF THE INVENTION
[0010] The present invention is based, in part, upon the
identification and characterization of a subset of DNA recognition
sequences that can act as efficient substrates for cleavage by the
rationally-designed, I-CreI-derived meganucleases (hereinafter,
"I-CreI-derived meganucleases").
[0011] In one aspect, the invention provides methods of identifying
sets of 22 base pair DNA sequences which can be cleaved by
I-CreI-derived meganucleases and which have, at their center, one
of a limited set of four base pair DNA center sequences that
contribute to more efficient cleavage by the I-CreI-derived
meganucleases. The invention also provides methods that use such
DNA sequences to produce recombinant nucleic acids, cells and
organisms by utilizing the recognition sequences as substrates for
I-CreI-derived meganucleases, and products incorporating such DNA
sequences.
[0012] Thus, in one aspect, the invention provides a method for
cleaving a double-stranded DNA comprising: (a) identifying in the
DNA at least one recognition site for a rationally-designed I
CreI-derived meganuclease with altered specificity relative to
I-CreI, wherein the recognition site is not cleaved by a
naturally-occurring I-CreI, wherein the recognition site has a four
base pair central sequence selected from the group consisting of
TTGT, TTAT, TCTT, TCGT, TCAT, GTTT, GTCT, GGAT, GAGT, GAAT, ATGT,
TTTC, TTCC, TGAC, TAAC, GTTC, ATAT, TCGA, TTAA, GGGC, ACGC, CCGC,
CTGC, ACAA, ATAA, AAGA, ACGA, ATGA, AAAC, AGAC, ATCC, ACTC, ATTC,
ACAT, GAAA, GGAA, GTCA, GTTA, GAAC, ATAT, TCGA, TTAA, GCCC, GCGT,
GCGG and GCAG; (b) providing the rationally-designed meganuclease;
and (c) contacting the DNA with the rationally-designed
meganuclease; whereby the rationally-designed meganuclease cleaves
the DNA.
[0013] In another aspect, the invention provides a method for
cleaving a double-stranded DNA comprising: (a) introducing into the
DNA a recognition site for a rationally-designed I CreI derived
meganuclease with altered specificity relative to I-CreI, wherein
the recognition site is not cleaved by a naturally-occurring
I-CreI, wherein the recognition site has a four base pair central
sequence selected from the group consisting of TTGT, TTAT, TCTT,
TCGT, TCAT, GTTT, GTCT, GGAT, GAGT, GAAT, ATGT, TTTC, TTCC, TGAC,
TAAC, GTTC, ATAT, TCGA, TTAA, GGGC, ACGC, CCGC, CTGC, ACAA, ATAA,
AAGA, ACGA, ATGA, AAAC, AGAC, ATCC, ACTC, ATTC, ACAT, GAAA, GGAA,
GTCA, GTTA, GAAC, ATAT, TCGA, TTAA, GCCC, GCGT, GCGG and GCAG; and
(b) providing the rationally-designed meganuclease; and (c)
contacting the DNA with the rationally-designed meganuclease;
whereby the rationally-designed meganuclease cleaves the DNA.
[0014] In some embodiments, the four base pair DNA sequence is
selected from the group consisting of GTGT, GTAT, TTAG, GTAG, TTAC,
TCTC, TCAC, GTCC, GTAC, TCGC, AAGC, GAGC, GCGC, GTGC, TAGC, TTGC,
ATGC, ACAC, ATAC, CTAA, CTAC, GTAA, GAGA, GTGA, GGAC, GTAC, GCGA,
GCTT, GCTC, GCGC, GCAC, GCTA, GCAA and GCAT.
[0015] In some embodiments, the DNA cleavage is in vitro. In other
embodiments, the DNA cleavage is in vivo.
[0016] In some embodiments, the DNA is selected from the group
consisting of a PCR product; an artificial chromosome; genomic DNA
isolated from bacteria, fungi, plants, or animal cells; and viral
DNA.
[0017] In some embodiments, the DNA is present in a cell selected
from the group consisting of a bacterial, fungal, plant and animal
cell.
[0018] In some embodiments, the DNA is present in a nucleic acid
selected from the group consisting of a plasmid, a prophage and a
chromosome.
[0019] In certain embodiments, the method further comprises
rationally-designing the I CreI derived meganuclease to recognize
the recognition site.
[0020] In some embodiments, the method further comprises producing
the rationally-designed I-CreI-derived meganuclease.
[0021] In another aspect, the invention provides a cell transformed
with a nucleic acid comprising, in order: a) a first 9 base pair
DNA sequence which can be bound by an I CreI derived meganuclease
monomer or by a first domain from a single-chain I CreI derived
meganuclease; b) a four base pair DNA sequence selected from the
group consisting of GTGT, GTAT, TTAG, GTAG, TTAC, TCTC, TCAC, GTCC,
GTAC, TCGC, AAGC, GAGC, GCGC, GTGC, TAGC, TTGC, ATGC, ACAC, ATAC,
CTAA, CTAC, GTAA, GAGA, GTGA, GGAC, GTAC, GCGA, GCTT, GCTC, GCGC,
GCAC, GCTA, GCAA and GCAT; and c) a second 9 base pair DNA sequence
which can be bound by an I CreI derived meganuclease monomer or by
a second domain from the single-chain I CreI derived meganuclease,
wherein the second 9 base pair DNA sequence is in the reverse
orientation relative to the first.
[0022] In yet another aspect, the invention provides a cell
containing an exogenous nucleic acid sequence integrated into its
genome, comprising, in order: a) a first exogenous 9 base pair DNA
sequence which can be bound by an I CreI derived meganuclease
monomer or by a first domain from a single-chain I CreI derived
meganuclease; b) an exogenous four base pair DNA sequence selected
from the group consisting of GTGT, GTAT, TTAG, GTAG, TTAC, TCTC,
TCAC, GTCC, GTAC, TCGC, AAGC, GAGC, GCGC, GTGC, TAGC, TTGC, ATGC,
ACAC, ATAC, CTAA, CTAC, GTAA, GAGA, GTGA, GGAC, GTAC, GCGA, GCTT,
GCTC, GCGC, GCAC, GCTA, GCAA and GCAT; and a) a second exogenous 9
base pair DNA sequence which can be bound by an I CreI derived
meganuclease monomer or by a second domain from the single-chain I
CreI derived meganuclease, wherein the second 9 base pair DNA
sequence is in the reverse orientation relative to the first.
[0023] In some embodiments, the nucleic acid is a plasmid, an
artificial chromosome, or a viral nucleic acid.
[0024] In some embodiments, the cell is a non-human animal cell, a
plant cell, a bacterial cell, or a fungal cell.
[0025] In some embodiments, the four base pair DNA sequence is
TTGT, TTAT, TCTT, TCGT, TCAT, GTTT, GTCT, GGAT, GAGT, GAAT, ATGT,
TTTC, TTCC, TGAC, TAAC, GTTC, ATAT, TCGA, TTAA, GGGC, ACGC, CCGC,
CTGC, ACAA, ATAA, AAGA, ACGA, ATGA, AAAC, AGAC, ATCC, ACTC, ATTC,
ACAT, GAAA, GGAA, GTCA, GTTA, GAAC, ATAT, TCGA, TTAA, GCCC, GCGT,
GCGG or GCAG.
[0026] In some embodiments, the four base pair DNA sequence is
GTGT, GTAT, TTAG, GTAG, TTAC, TCTC, TCAC, GTCC, GTAC, TCGC, AAGC,
GAGC, GCGC, GTGC, TAGC, TTGC, ATGC, ACAC, ATAC, CTAA, CTAC, GTAA,
GAGA, GTGA, GGAC, GTAC, GCGA, GCTT, GCTC, GCGC, GCAC, GCTA, GCAA or
GCAT.
[0027] In yet another aspect, the invention provides a method of
conducting a custom-designed, I-CreI-derived meganuclease business
comprising: (a) receiving a DNA sequence into which a double-strand
break is to be introduced by a rationally-designed I CreI-derived
meganuclease; (b) identifying in the DNA sequence at least one
recognition site for a rationally-designed I CreI-derived
meganuclease with altered specificity relative to I-CreI, wherein
the recognition site is not cleaved by a naturally-occurring
I-CreI, wherein the recognition site has a four base pair central
sequence selected from the group consisting of TTGT, TTAT, TCTT,
TCGT, TCAT, GTTT, GTCT, GGAT, GAGT, GAAT, ATGT, TTTC, TTCC, TGAC,
TAAC, GTTC, ATAT, TCGA, TTAA, GGGC, ACGC, CCGC, CTGC, ACAA, ATAA,
AAGA, ACGA, ATGA, AAAC, AGAC, ATCC, ACTC, ATTC, ACAT, GAAA, GGAA,
GTCA, GTTA, GAAC, ATAT, TCGA, TTAA, GCCC, GCGT, GCGG and GCAG; and
(c) providing the rationally-designed meganuclease.
[0028] In some embodiments, the method further comprises
rationally-designing the I CreI derived meganuclease to recognize
the recognition site.
[0029] In some embodiments, the method further comprises producing
the rationally-designed meganuclease.
[0030] In some embodiments, the rationally-designed meganuclease is
provided to the same party from which the DNA sequence has been
received.
[0031] These and other aspects and embodiments of the invention
will be apparent to one of ordinary skill in the art from the
following detailed description of the invention, figures and
appended claims.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1. (A) Schematic illustration of the interactions
between the naturally-occurring I-CreI homodimer and a
double-stranded recognition sequence, based upon crystallographic
data. This schematic representation depicts one recognition
sequence (SEQ ID NO: 2 and SEQ ID NO: 3), shown as unwound for
illustration purposes only, bound by the homodimer, shown as two
ovals. The bases of each DNA half-site are numbered -1 through -9,
and the amino acid residues of I-CreI which form the recognition
surface are indicated by one-letter amino acid designations and
numbers indicating residue position. The four base pairs that
comprise the center sequence are numbered +1 to +4. Solid black
lines: hydrogen bonds to DNA bases. (B) One wild-type I-CreI
recognition sequence (SEQ ID NO: 2 and SEQ ID NO: 3) showing the
locations of the inverted half-sites and center sequence.
[0033] FIG. 2. (A) Schematic diagram of the plasmid substrates
evaluated to determine center sequence preference. A set of pUC-19
plasmids were produced which harbored potential recognition
sequences for the genetically-engineered meganuclease DJ1. These
potential recognition sequences comprised a pair of inverted DJ1
half-sites separated by a variety of different four base pair
center sequences (numbered +1 through +4), as described below. (B)
Example of gel electrophoresis data showing DJ1 meganuclease
cleavage of plasmid substrates described in (A). The "uncut" arrow
indicates XmnI linearized plasmid substrate. The "cut" arrows
indicate XmnI linearized plasmid substrates which have also been
successfully cleaved by DJ1.
[0034] FIG. 3. (A) Schematic diagram of a T-DNA that was stably
integrated into the Arabidopsis thaliana genome as described in
Example 1. In this T-DNA construct, a codon-optimized gene encoding
the genetically-engineered BRP2 meganuclease (BRP2) (SEQ ID NO: 8)
is under the control of a Hsp70 promoter (HSP) and a NOS terminator
(TERM). A pair of potential BRP2 recognition sequences (Site1,
Site2) are housed adjacent to the terminator separated by 7 base
pairs containing a PstI restriction enzyme site (PstI). A kanamycin
resistance marker (Kan) is also housed on the T-DNA to allow
selection for stable transformants. (B) The expected product
following BRP2 meganuclease cleavage of Site1 and Site2 showing
loss of the intervening 7 base pair fragment and PstI restriction
site. Arrows show the location of PCR primers used to screen for
cleavage of the T-DNA. (C) Sequences of the BRP2 recognition
sequences housed on either the GTAC construct (GTAC) or the TAGA
construct (TAGA) with center sequences underlined. (D) Example of
electrophoresis data from a plant transformed with the GTAC
construct. Genomic DNA was isolated from the leaves of Arabidopsis
seedlings stably transformed with either the GTAC T-DNA construct
before and after a 2 hour "heat-shock" to induce BRP2 expression.
DNA samples were then added to PCR reactions using the primers
shown in (B). PCR reactions were digested with PstI and visualized
by gel electrophoresis. C: control lane lacking PstI. 44, 45, and
46: PCR samples from three representative plants showing nearly
complete digestion by PstI in samples taken prior to heat shock
(-lanes) and very little digestion by PstI in samples taken after
heat-shock (+lanes). These results indicate that the BRP2
meganuclease was able to cleave the BRP2 recognition sequence which
incorporated a GTAC center sequence in vivo.
[0035] FIG. 4. (A) Schematic diagram of a T-DNA that was stably
integrated into the Arabidopsis thaliana genome as described in
Example 2. In this T-DNA construct, a codon-optimized gene encoding
the BRP12-SC meganuclease (BRP12-SC) (SEQ ID NO: 15) is under the
control of a Hsp70 promoter (HSP) and a NOS terminator (TERM). A
pair of potential BRP12-SC recognition sequences (Site1, Site2) are
housed adjacent to the terminator separated by 7 base pairs
containing a PstI restriction enzyme site (PstI). A kanamycin
resistance marker (Kan) is also housed on the T-DNA to allow
selection for stable transformants. (B) The expected product
following BRP12-SC meganuclease cleavage of Site1 and Site2 showing
loss of the intervening 7 base pair fragment and PstI restriction
site. Arrows show the location of PCR primers used to screen for
cleavage of the T-DNA. (C) Sequences of the BRP12-SC recognition
sequences housed on either the GTAC construct (GTAC) or the TAGA
construct (TAGA) with center sequences underlined.
[0036] FIG. 5. Graphic representation of the effects of
meganuclease concentration and center sequence on in vitro
meganuclease cleavage. The BRP2 meganuclease (SEQ ID NO: 8, see
Example 1) was added at the indicated concentration to a digest
reaction containing 0.25 picomoles of a plasmid substrate harboring
either a BRP2 recognition sequence with the center sequence GTAC or
a BRP2 recognition sequence with the center sequence TAGA.
Reactions were 25 microliters in SA buffer (25 mM Tris-HCL, pH 8.0,
100 mM NaCl, 5 mM MgCl.sub.2, 5 mM EDTA). Reactions were incubated
at 37.degree. C. for 2 hours and were then visualized by gel
electrophoresis and the percent of plasmid substrate cleaved by the
meganuclease was plotted as a function of meganuclease
concentration.
DETAILED DESCRIPTION OF THE INVENTION
1.1 Introduction
[0037] The present invention is based, in part, upon the
identification and characterization of particular DNA sequences
that are more efficiently cleaved by the rationally-designed,
I-CreI-derived meganucleases. Specifically, the invention is based
on the discovery that certain four-base pair DNA sequences, when
incorporated as the central four-base pairs of a
rationally-designed, I-CreI-derived meganuclease recognition
sequence, can significantly impact cleavage by the corresponding
meganuclease although the meganuclease does not, based upon
analysis of crystal structures, appear to contact the central four
base pairs. As there are four DNA bases (A, C, G, and T), there are
4.sup.4 or 256 possible DNA sequences that are four base pairs in
length. All of these possible sequences were examined to determine
the subsets of sequences that are more efficiently cleaved by
I-CreI-derived meganucleases. The results of this analysis allow
for more accurate prediction of whether or not a particular
double-stranded DNA site 22 base pairs in length can be more
efficiently cleaved by the I-CreI-derived meganuclease.
1.2 References and Definitions
[0038] The patent and scientific literature referred to herein
establishes knowledge that is available to those of skill in the
art. The issued U.S. patents, allowed applications, published
foreign applications, and references, including GenBank database
sequences, that are cited herein are hereby incorporated by
reference to the same extent as if each was specifically and
individually indicated to be incorporated by reference.
[0039] As used herein, the term "I-CreI-derived meganuclease"
refers to a rationally-designed (i.e., genetically-engineered)
meganuclease that is derived from I-CreI. The term
genetically-engineered meganuclease, as used herein, refers to a
recombinant variant of an I-CreI homing endonuclease that has been
modified by one or more amino acid insertions, deletions or
substitutions that affect one or more of DNA-binding specificity,
DNA cleavage activity, DNA-binding affinity, and/or dimerization
properties. Some genetically-engineered meganucleases are known in
the art (see, e.g., Porteus et al. (2005), Nat. Biotechnol. 23:
967-73; Sussman et al. (2004), J. Mol. Biol. 342: 31-41; Epinat et
al. (2003), Nucleic Acids Res. 31: 2952-62) and a general method
for rationally-designing such variants is disclosed in WO
2007/047859. Additional methods for genetically-engineering such
variants are disclosed in WO 04/067736, WO 07/060495, WO 06/097853,
WO 07/049095, WO 08/102198, WO 08/010093, WO 08/010009, WO
07/093918, WO 07/093836, WO 08/102274, WO 08/059317, WO 09/013622,
WO 09/019614, WO 09/019528, WO 08/152523, WO 04/067753, WO
03/078619, WO 06/097784, WO 07/034262, WO 07/049156, WO 07/057781,
WO 08/093152, WO 08/102199, WO 08/102274, WO 08/149176, WO
09/013559, WO 09/013622, and WO 09/019528.
[0040] A meganuclease may bind to double-stranded DNA as a
homodimer, as is the case for wild-type I-CreI, or it may bind to
DNA as a heterodimer. A meganuclease may also be a "single-chain
heterodimer" in which a pair of DNA-binding domains derived from
I-CreI are joined into a single polypeptide using a peptide linker
The term "homing endonuclease" is synonymous with the term
"meganuclease."
[0041] As used herein, the term "rationally-designed" means
non-naturally occurring and/or genetically engineered. The
rationally-designed meganucleases of the invention differ from
wild-type or naturally-occurring meganucleases in their amino acid
sequence or primary structure, and may also differ in their
secondary, tertiary or quaternary structure. In addition, the
rationally-designed meganucleases of the invention also differ from
wild-type or naturally-occurring meganucleases in recognition
sequence-specificity and/or activity.
[0042] As used herein, with respect to a protein, the term
"recombinant" means having an altered amino acid sequence as a
result of the application of genetic engineering techniques to
nucleic acids which encode the protein, and cells or organisms
which express the protein. With respect to a nucleic acid, the term
"recombinant" means having an altered nucleic acid sequence as a
result of the application of genetic engineering techniques.
Genetic engineering techniques include, but are not limited to, PCR
and DNA cloning technologies; transfection, transformation and
other gene transfer technologies; homologous recombination;
site-directed mutagenesis; and gene fusion. In accordance with this
definition, a protein having an amino acid sequence identical to a
naturally-occurring protein, but produced by cloning and expression
in a heterologous host, is not considered recombinant.
[0043] As used herein, the term "genetically-modified" refers to a
cell or organism in which, or in an ancestor of which, a genomic
DNA sequence has been deliberately modified by recombinant
technology. As used herein, the term "genetically-modified"
encompasses the term "transgenic."
[0044] As used herein, the term "wild-type" refers to any
naturally-occurring form of a meganuclease. The term "wild-type" is
not intended to mean the most common allelic variant of the enzyme
in nature but, rather, any allelic variant found in nature.
Wild-type meganucleases are distinguished from recombinant or
non-naturally-occurring meganucleases.
[0045] As used herein, the term "recognition sequence half-site" or
simply "half site" means a 9 base pair DNA sequence which is
recognized by a meganuclease monomer, in the case of a dimeric
meganuclease, or by one domain of a single-chain meganuclease.
[0046] As used herein, the term "recognition sequence" refers to a
pair of half-sites which is bound and cleaved by a meganuclease. A
recognition sequence comprises a pair of inverted, 9 base pair half
sites separated by four base-pairs. The recognition sequence is,
therefore, 22 base-pairs in length. The base pairs of each
half-site are designated -9 through -1, with the -9 position being
most distal from the cleavage site and the -1 position being
adjacent to the 4 base pair center sequence, the base pairs of
which are designated +1 through +4. The strand of each half-site
which is oriented 5' to 3' in the direction from -9 to -1 (i.e.,
towards the cleavage site), is designated the "sense" strand, and
the opposite strand is designated the "antisense strand", although
neither strand may encode protein. Thus, the "sense" strand of one
half-site is the antisense (opposite) strand of the other
half-site. See, for example, FIG. 1a.
[0047] As used herein, the term "center sequence" refers to the
four base pairs separating half sites in the meganuclease
recognition sequence. These bases are numbered +1 through +4 in
FIG. 1a. The center sequence comprises the four bases that become
the 3' single-strand overhangs following meganuclease cleavage.
"Center sequence" can refer to the sequence of the sense strand or
the antisense (opposite) strand.
[0048] As used herein, the term "specificity" refers to the ability
of a meganuclease to recognize and cleave double-stranded DNA
molecules only at a particular subset of all possible recognition
sequences. The set of recognition sequences will share certain
conserved positions or sequence motifs, but may be degenerate at
one or more positions. A more specific meganuclease is capable of
binding and cleaving a smaller subset of the possible recognition
sequences, whereas a less specific meganuclease is capable of
binding and cleaving a larger subset of the possible recognition
sequences.
[0049] As used herein, the term "palindromic" refers to a
recognition sequence consisting of inverted repeats of identical
half-sites. In this case, however, the palindromic sequence need
not be palindromic with respect to the center sequence, which is
not contacted by the enzyme. In the case of dimeric meganucleases,
palindromic DNA sequences are recognized by homodimers in which the
two monomers make contacts with identical half-sites.
[0050] As used herein, the term "pseudo-palindromic" refers to a
recognition sequence consisting of inverted repeats of
non-identical or imperfectly palindromic half-sites. In this case,
the pseudo-palindromic sequence need not be palindromic with
respect to the center sequence, and also can deviate from a
perfectly palindromic sequence between the two half-sites.
Pseudo-palindromic DNA sequences are typical of the natural DNA
sites recognized by wild-type homodimeric meganucleases in which
two identical enzyme monomers make contacts with different
half-sites.
[0051] As used herein, the term "non-palindromic" refers to a
recognition sequence composed of two unrelated half-sites of a
meganuclease. In this case, the non-palindromic sequence need not
be palindromic with respect to either the center sequence or the
two monomer half-sites. Non-palindromic DNA sequences are
recognized by either heterodimeric meganucleases or single-chain
meganucleases comprising a pair of domains that recognize
non-identical half-sites.
[0052] As used herein, the term "activity" refers to the rate at
which a meganuclease of the invention cleaves a particular
recognition sequence. Such activity is a measurable enzymatic
reaction, involving the hydrolysis of phosphodiester bonds of
double-stranded DNA. The activity of a meganuclease acting on a
particular DNA substrate is affected by the affinity or avidity of
the meganuclease for that particular DNA substrate which is, in
turn, affected by both sequence-specific and non-sequence-specific
interactions with the DNA.
[0053] As used herein, the term "homologous recombination" refers
to the natural, cellular process in which a double-stranded
DNA-break is repaired using a homologous DNA sequence as the repair
template (see, e.g., Cahill et al. (2006), Front. Biosci.
11:1958-1976). The homologous DNA sequence may be an endogenous
chromosomal sequence or an exogenous nucleic acid that was
delivered to the cell. Thus, for some applications, a meganuclease
is used to cleave a recognition sequence within a target sequence
in a genome and an exogenous nucleic acid with homology to or
substantial sequence similarity with the target sequence is
delivered into the cell and used as a template for repair by
homologous recombination. The DNA sequence of the exogenous nucleic
acid, which may differ significantly from the target sequence, is
thereby incorporated into the chromosomal sequence. The process of
homologous recombination occurs primarily in eukaryotic organisms.
The term "homology" is used herein as equivalent to "sequence
similarity" and is not intended to require identity by descent or
phylogenetic relatedness.
[0054] As used herein, the term "non-homologous end-joining" refers
to the natural, cellular process in which a double-stranded
DNA-break is repaired by the direct joining of two non-homologous
DNA segments (see, e.g., Cahill et al. (2006), Front. Biosci.
11:1958-1976). DNA repair by non-homologous end joining is
error-prone and frequently results in the untemplated addition or
deletion of DNA sequences at the site of repair. Thus, for some
applications, a meganuclease can be used to produce a
double-stranded break at a meganuclease recognition sequence within
a target sequence in a genome to disrupt a gene (e.g., by
introducing base insertions, base deletions, or frameshift
mutations) by non-homologous end joining For other applications, an
exogenous nucleic acid lacking homology to or substantial sequence
similarity with the target sequence may be captured at the site of
a meganuclease-stimulated double-stranded DNA break by
non-homologous end joining (see, e.g., Salomon et al. (1998), EMBO
J. 17:6086-6095). The process of non-homologous end joining occurs
in both eukaryotes and prokaryotes such as bacteria.
[0055] As used herein, the term "sequence of interest" means any
nucleic acid sequence, whether it codes for a protein, RNA, or
regulatory element (e.g., an enhancer, silencer, or promoter
sequence), that can be inserted into a genome or used to replace a
genomic DNA sequence using a meganuclease protein. Sequences of
interest can have heterologous DNA sequences that allow for tagging
a protein or RNA that is expressed from the sequence of interest.
For instance, a protein can be tagged with tags including, but not
limited to, an epitope (e.g., c-myc, FLAG) or other ligand (e.g.,
poly-His). Furthermore, a sequence of interest can encode a fusion
protein, according to techniques known in the art (see, e.g.,
Ausubel et al., Current Protocols in Molecular Biology, Wiley
1999). For some applications, the sequence of interest is flanked
by a DNA sequence that is recognized by the meganuclease for
cleavage. Thus, the flanking sequences are cleaved allowing for
proper insertion of the sequence of interest into genomic
recognition sequences cleaved by a meganuclease. For some
applications, the entire sequence of interest is homologous to or
has substantial sequence similarity with a target sequence in the
genome such that homologous recombination effectively replaces the
target sequence with the sequence of interest. For other
applications, the sequence of interest is flanked by DNA sequences
with homology to or substantial sequence similarity with the target
sequence such that homologous recombination inserts the sequence of
interest within the genome at the locus of the target sequence. For
some applications, the sequence of interest is substantially
identical to the target sequence except for mutations or other
modifications in the meganuclease recognition sequence such that
the meganuclease can not cleave the target sequence after it has
been modified by the sequence of interest.
[0056] As used herein, the term "single-chain meganuclease" refers
to a polypeptide comprising a pair of meganuclease subunits joined
by a linker. A single-chain meganuclease has the organization:
N-terminal subunit--Linker--C-terminal subunit. The two
meganuclease subunits, each of which is derived from I-CreI, will
generally be non-identical in amino acid sequence and will
recognize non-identical half-sites. Thus, single-chain
meganucleases typically cleave pseudo-palindromic or
non-palindromic recognition sequences. A single chain meganuclease
may be referred to as a "single-chain heterodimer" or "single-chain
heterodimeric meganuclease" although it is not, in fact,
dimeric.
[0057] As used herein, unless specifically indicated otherwise, the
word "or" is used in the inclusive sense of "and/or" and not the
exclusive sense of "either/or."
2.1 Preferred Center Sequences for I-CreI-Derived Meganucleases
[0058] The present invention is based, in part, in the
identification of subsets of the possible four base pair center
sequences that are preferred by I-CreI-derived meganucleases. As
the wild-type enzyme does not make significant contacts with the
bases in the center sequence, the same center sequence preferences
of the wild-type I-CreI homing nuclease apply to
rationally-designed I-CreI-derived meganucleases which have been
redesigned with respect to, for example, half-site preference,
DNA-binding affinity, and/or heterodimerization ability. This
invention provides, therefore, important criteria that can be
considered in determining whether or not a particular 22 base pair
DNA sequence is a suitable I-CreI-derived meganuclease recognition
sequence.
[0059] The preferred set of center sequences was determined using a
genetically-engineered meganuclease called "DJ1" (SEQ ID NO: 4).
The production of this meganuclease is described in WO 2007/047859.
DJ1 is a homodimeric I-CreI-derived meganuclease which was designed
to recognize a palindromic meganuclease recognition sequence (SEQ
ID NO: 5, SEQ ID NO: 6) that differs at 4 positions per half-site
relative to wild-type I-CreI. This change in half-site specificity
was achieved by the introduction of 6 amino acid substitutions to
wild type I-CreI (K28D, N30R, S32N, Q38E, S40R, and T42R).
[0060] To test for cleavage activity with respect to various
recognition sequences, DJ1 was expressed in E. coli and purified as
described in Example 1 of WO 2007/047859. Then, 25 picomoles of
purified meganuclease protein were added to a 10 nM solution of
plasmid DNA substrate in SA buffer (25 mM Tris-HCL, pH 8.0, 100 mM
NaCl, 5 mM MgCl.sub.2, 5 mM EDTA) in a 25 microliter reaction. 1
microliter of XmnI restriction enzyme was added to linearize the
plasmid substrates. Reactions were incubated at 37.degree. C. for 4
hours and were then visualized by gel electrophoresis to determine
the extent to which each was cleaved by the DJ1 meganuclease.
[0061] The plasmid substrates used in these experiments comprised a
pUC-19 plasmid in which a potential meganuclease recognition
sequence was inserted into the polylinker site (SmaI site). Each
potential meganuclease recognition site comprised a pair of
inverted DJ1 half-sites (SEQ ID NO: 7) separated by a different
center sequence. Thus, by evaluating DJ1 cleavage of multiple DNA
substrates differing only by center sequence, it was possible to
determine which center sequences are the most amenable to
meganuclease cleavage (FIG. 2).
[0062] Initially, only the influence of the N.sub.+2 and N.sub.+3
bases were evaluated. The X-ray crystal structure of I-CreI in
complex with its natural DNA site shows that the DNA is distorted
at these central two base pairs (Jurica et al. (1998), Mol Cell.
2:469-76). Computer modeling suggests that a purine (G or A) at
N.sub.+2 is incompatible with a pyrimidine (C or T) at N.sub.+3.
This is because the distortion introduced by I-CreI binding causes
a steric clash between a purine base at N.sub.+2 and a second
purine base-paired to a pyrimidine at N.sub.+3. This expected
incompatibility was verified experimentally by incubating DJ1
protein with plasmid substrates harboring meganuclease recognition
sites with all possible center sequences of the form
A.sub.+1X.sub.+2X.sub.-3T.sub.+4 in which X is any base. The
results are summarized in Table 1. For Tables 1-5, "Activity"
refers to the following:
[0063] -: no cleavage in 4 hours
[0064] +: 1%-25% cleavage in 4 hours
[0065] ++: 26%-75% cleavage in 4 hours
[0066] +++: 75%-100% cleavage in 4 hours
TABLE-US-00001 TABLE 1 The effect of changes at N.sub.+2 and
N.sub.+3 Seq. No. N.sub.+1 N.sub.+2 N.sub.+3 N.sub.+4 Activity 1 A
A A T + 2 A A C T - 3 A A G T + 4 A A T T - 5 A C A T + 6 A C C T +
7 A C G T + 8 A C T T + 9 A G A T + 10 A G C T - 11 A G G T + 12 A
G T T - 13 A T A T ++ 14 A T C T + 15 A T G T ++ 16 A T T T +
[0067] Consistent with the computer modeling, it was found that the
four plasmid substrates with a purine base at N.sub.+2 and a
pyrimidine base at N.sub.+3 (sequence numbers 2, 4, 10, and 12)
were not cut efficiently by DJ1.
[0068] Next, a more comprehensive evaluation of center sequence
preference was performed. There are 4.sup.4 or 256 possible center
sequences. Of these, 25%, or 64, have a purine base at N.sub.+2 and
pyrimidine at N.sub.+3 and, therefore, were eliminated as center
sequences based on the experiment described above. Of the remaining
192, 92 are redundant because meganucleases are symmetric and
recognize bases equally on both the sense and antisense strand. For
example, the sequence A.sub.+1A.sub.+2A.sub.+3A.sub.+4 on the sense
strand is recognized by the meganuclease as
T.sub.+1T.sub.-2T.sub.+3T.sub.+4 on the antisense strand and, thus,
A.sub.+1A.sub.+2A.sub.+3A.sub.+4 and
T.sub.+1T.sub.+2T.sub.+3T.sub.+4 are functionally equivalent.
Taking these redundancies into account, as well as the
aforementioned N.sub.+2/N.sub.+3 conflicts, there were 100 possible
center sequences remaining. To determine which of these were
preferred by meganucleases, we produced 100 plasmid substrates
harboring these 100 center sequences flanked by inverted
recognition half-sites for the DJ1 meganuclease. DJ1 was then
incubated with each of the 100 plasmids and cleavage activity was
evaluated as described above. These results are summarized in Table
2.
TABLE-US-00002 TABLE 2 Cleavable Center Sequences Seq. No. N.sub.+1
N.sub.+2 N.sub.+3 N.sub.+4 Activity 1 T T T T + 2 T T G T ++ 3 T T
C T + 4 T T A T ++ 5 T G G T + 6 T G A T + 7 T C T T ++ 8 T C G T
++ 9 T C C T + 10 T C A T ++ 11 T A G T + 12 T A A T + 13 G T T T
++ 14 G T G T +++ 15 G T C T ++ 16 G T A T +++ 17 G G G T + 18 G G
A T ++ 19 G A G T ++ 20 G A A T ++ 21 C T T T + 22 C T G T + 23 C T
C T + 24 C T A T + 25 C G G T + 26 C G A T + 27 C C T T + 28 C C G
T + 29 C C C T + 30 C C A T + 31 C A G T + 32 C A A T + 33 A T T T
+ 34 A T G T ++ 35 A T C T + 36 A G G T + 37 A C T T + 38 T T T G +
39 T T G G + 40 T T C G + 41 T T A G +++ 42 T G G G + 43 T G A G +
44 T C T G + 45 T C G G + 46 T C C G + 47 T C A G + 48 T A G G + 49
T A A G + 50 G T T G + 51 G T G G + 52 G T C G + 53 G T A G +++ 54
G G G G + 55 G G A G + 56 G A G G + 57 G A A G + 58 C T T G + 59 C
T G G + 60 C T C G + 61 C G G G + 62 C C T G + 63 T T T C ++ 64 T T
C C ++ 65 T T A C +++ 66 T G A C ++ 67 T C T C +++ 68 T C C C + 69
T C A C +++ 70 T A A C ++ 71 G T T C ++ 72 G T C C +++ 73 T T T A +
74 T T G A + 75 T T C A + 76 T G G A + 77 T C T A + 78 A T A T ++
79 A C G T + 80 C T A G + 81 C C G G + 82 G T A C +++ 83 T C G A ++
84 T T A A ++ 85 T C G C +++ 86 A A G C +++ 87 G A G C +++ 88 G C G
C +++ 89 G G G C ++ 90 G T G C +++ 91 T A G C +++ 92 T G G C + 93 T
T G C +++ 94 A C G C ++ 95 A G G C + 96 A T G C +++ 97 C A G C + 98
C C G C ++ 99 C G G C + 100 C T G C +++
[0069] For clarity, each of the center sequences listed in Table 2
is equivalent to its opposite strand sequence due to the fact that
the I-CreI meganuclease binds its recognition sequence as a
symmetric homodimer. Thus, sequence no. 100 in Table 2,
C.sub.+1T.sub.+2G.sub.+3C.sub.+4, is equivalent to its opposite
strand sequence, G.sub.+1C.sub.+2A.sub.+3G.sub.+4. From these data,
a general set of center sequence preference rules emerge. These
rules, which are not meant to supersede Table 1 or Table 2,
include: [0070] 1. Center sequences with a purine base at N.sub.+2
and a pyrimidine base at N.sub.+3 cut very poorly, if at all.
[0071] 2. G is preferred at N.sub.+1. This is equivalent to C at
N.sub.+4. All of the most preferred center sequences have G at
N.sub.+1 and/or C at N.sub.+4. [0072] 3. C is preferred at
N.sub.+2. This is equivalent to G at N.sub.+3. [0073] 4. There is a
preference for center sequences with a pyrimidine base at N.sub.+2
and a purine base at N.sub.+3. [0074] 5. There is a preference for
sequences with at least 1 A-T base pair in the center sequence.
[0075] Thus, in general, preferred center sequences have the form
G.sub.+N.sub.+2R.sub.+3X.sub.+4 where Y is a pyrimidine (C or T), R
is a purine (A or G), and X is any base (A, C, G, or T).
2.2 In Vitro Applications Using Preferred Center Sequences.
[0076] Genetically-engineered meganucleases have numerous potential
in vitro applications including restriction mapping and cloning.
These applications are known in the art and are discussed in WO
2007/047859.
[0077] One advantage of using genetically-engineered meganucleases
rather than conventional restriction enzymes for applications such
as cloning is the possibility of cutting DNA to leave a wide range
of different 3' overhangs ("sticky ends") that are compatible with,
for example, the 3' overhangs produced by cleaving a particular
vector of interest. Thus, there are occasions when it is desirable
to cleave a meganuclease recognition sequence with a sub-optimal
center sequence in order to create a desired overhang.
[0078] Because in vitro DNA cleavage conditions are, in general,
less stringent than conditions in vivo, the use of sub-optimal
center sequences may be acceptable for such applications. For
example, relative to in vivo applications, in vitro digests using
engineered meganucleases can be performed at a higher ratio of
meganuclease to DNA, there is typically less non-specific (genomic)
DNA competing for meganuclease, and solution conditions can be
optimized to favor meganuclease cleavage (e.g., using SA buffer as
described above). Thus, a larger number of center sequences are
suitable for in vitro applications than for in vivo applications.
All of the center sequences listed in Table 2 are suitable for in
vitro applications, but preferred and most preferred center
sequences for in vitro applications are listed in Table 3 and Table
4, respectively, with their opposite strand sequences.
TABLE-US-00003 TABLE 3 Preferred Center Sequences for in vitro
Applications Seq. Opposite Strand No.
N.sub.+1N.sub.+2N.sub.+3N.sub.+4 Sequence 1 TTGT ACAA 2 TTAT ATAA 3
TCTT AAGA 4 TCGT ACGA 5 TCAT ATGA 6 GTTT AAAC 7 GTCT AGAC 8 GGAT
ATCC 9 GAGT ACTC 10 GAAT ATTC 11 ATGT ACAT 12 TTTC GAAA 13 TTCC
GGAA 14 TGAC GTCA 15 TAAC GTTA 16 GTTC GAAC 17 ATAT ATAT 18 TCGA
TCGA 19 TTAA TTAA 20 GGGC GCCC 21 ACGC GCGT 22 CCGC GCGG 23 CTGC
GCAG
TABLE-US-00004 TABLE 4 Most Preferred Center Sequences for in vitro
Applications Seq. Opposite Strand No. N.sub.1N.sub.2N.sub.3N.sub.4
Sequence 1 GTGT ACAC 2 GTAT ATAC 3 TTAG CTAA 4 GTAG CTAC 5 TTAC
GTAA 6 TCTC GAGA 7 TCAC GTGA 8 GTCC GGAC 9 GTAC GTAC 10 TCGC GCGA
11 AAGC GCTT 12 GAGC GCTC 13 GCGC GCGC 14 GTGC GCAC 15 TAGC GCTA 16
TTGC GCAA 17 ATGC GCAT
[0079] Obviously, not every 22 base pair DNA sequence having a
preferred or most preferred center sequence is capable of being a
meganuclease recognition sequence in vitro. The sequence of the
half-sites flanking the center sequence must also be amenable to
meganuclease recognition and cleavage. Methods for engineering a
meganuclease including I-CreI, to recognize a pre-determined
half-site are known in the art (see, e.g., WO 2007/047859). Thus, a
preferred I-CreI-derived meganuclease recognition sequence for in
vitro applications will comprise: (1) a first 9 base pair half-site
amenable to recognition by a meganuclease monomer (or a first
domain of a single-chain meganuclease); (2) a preferred or most
preferred center sequence from Table 2 or Table 3; and (3) a second
9 base pair half-site amenable to recognition by a meganuclease
monomer (or a second domain of a single-chain meganuclease) in the
reverse orientation relative to the first half-site.
[0080] Thus, in one aspect, the invention provides methods for
cleaving a double-stranded DNA in vitro by (a) identifying at least
one potential recognition site for at least one I-CreI-derived
meganuclease within the DNA, wherein the potential recognition site
has a four base pair central sequence selected from the group of
central sequences of Table 2; (b) identifying an I-CreI-derived
meganuclease which recognizes that recognition site in the DNA; and
(c) contacting the I-CreI-derived meganuclease with the DNA;
whereby the I-CreI meganuclease cleaves the DNA.
[0081] In another aspect, the invention provides methods for
cleaving a double-stranded DNA in vitro by (a) introducing into the
DNA a recognition site for an I-CreI-derived meganuclease having a
four base pair central sequence selected from the group consisting
of central sequences of Table 2; and (b) contacting the
I-CreI-derived meganuclease with the DNA; whereby the
I-CreI-derived meganuclease cleaves the DNA.
[0082] In particular, in some embodiments, the DNA is selected from
a PCR product; an artificial chromosome; genomic DNA isolated from
bacteria, fungi, plants, or animal cells; and viral DNA.
[0083] In some embodiments, the DNA is present in a nucleic acid
selected from a plasmid, a prophage and a chromosome.
[0084] In some of the foregoing embodiments, the four base pair DNA
sequence is selected from Table 3. In other embodiments, the four
base pair DNA sequence is selected from Table 4.
[0085] In some embodiments, the I-CreI-derived meganuclease can be
specifically designed for use with the chosen recognition site in
the method.
2.3 In vivo Applications Using Preferred Center Sequences.
[0086] Applications such as gene therapy, cell engineering, and
plant engineering require meganuclease function inside of a living
cell (for clarity, any intracellular application will be referred
to as an "in vivo" application whether or not such cell is isolated
or part of a multicellular organism). These applications are known
in the art and are described in, e.g., WO 2007/047859. In vivo
applications are significantly restricted relative to in vitro
applications with regard to the center sequence. This is because
intracellular conditions cannot be manipulated to any great extent
to favor meganuclease activity and/or because vast amounts of
genomic DNA compete for meganuclease binding. Thus, only
meganuclease recognition sequences with optimal center sequences
are preferred for in vivo applications. Such sequences are listed
in Table 5 with their opposite strand sequences.
TABLE-US-00005 TABLE 5 Preferred Center Sequences for in vivo
applications. Seq. Opposite Strand No. N.sub.1N.sub.2N.sub.3N.sub.4
Sequence 1 GTGT ACAC 2 GTAT ATAC 3 TTAG CTAA 4 GTAG CTAC 5 TTAC
GTAA 6 TCTC GAGA 7 TCAC GTGA 8 GTCC GGAC 9 GTAC GTAC 10 TCGC GCGA
11 AAGC GCTT 12 GAGC GCTC 13 GCGC GCGC 14 GTGC GCAC 15 TAGC GCTA 16
TTGC GCAA 17 ATGC GCAT
[0087] Obviously, not every 22 base pair DNA sequence having a
preferred center sequence is capable of being a meganuclease
recognition sequence in vivo. The sequence of the half-sites
flanking the center sequence must also be amenable to meganuclease
recognition and cleavage. Methods for engineering a meganuclease,
including I-CreI, to recognize a pre-determined half-site are known
in the art (see, e.g., WO 2007/047859). Thus, a preferred in vivo
meganuclease recognition sequence will comprise: (1) a first 9 base
pair half-site amenable to recognition by a meganuclease monomer
(or a first domain of a single-chain meganuclease); (2) a preferred
center sequence from Table 5; and (3) a second 9 base pair
half-site amenable to recognition by a meganuclease monomer (or a
second domain of a single-chain meganuclease) in the reverse
orientation relative to the first half-site.
[0088] Thus, in one aspect, the invention provides methods for
cleaving a double-stranded DNA in vivo by (a) identifying at least
one potential recognition site for at least one I-CreI-derived
meganuclease within the DNA, wherein the potential recognition site
has a four base pair central sequence selected from the group of
central sequences of Table 2; (b) identifying an I-CreI-derived
meganuclease which recognizes that recognition site in the DNA; and
(c) contacting the I-CreI-derived meganuclease with the DNA;
whereby the I-CreI-derived meganuclease cleaves the DNA.
[0089] In another aspect, the invention provides methods for
cleaving a double-stranded DNA in vivo by (a) introducing into the
DNA a recognition site for an I-CreI-derived meganuclease having a
four base pair central sequence selected from the group consisting
of central sequences of Table 2; and (b) contacting the
I-CreI-derived meganuclease with the DNA; whereby the
I-CreI-derived meganuclease cleaves the DNA.
[0090] In some embodiments, the DNA is present in a cell selected
from a bacterial, fungal, plant and animal cell.
[0091] In some embodiments, the DNA is present in a nucleic acid
selected from a plasmid, a prophage and a chromosome.
[0092] In some of the foregoing embodiments, the four base pair DNA
sequence is selected from Table 3. In other embodiments, the four
base pair DNA sequence is selected from Table 4.
[0093] In some embodiments, the I-CreI-derived meganuclease is
specifically designed for use with the chosen recognition site in
the methods of the invention.
[0094] In some of the foregoing embodiments, the method includes
the additional step of rationally-designing the I-CreI-derived
meganuclease to recognize the chosen recognition site. In some
embodiments, the method further comprises producing the
I-CreI-derived meganuclease.
[0095] In another aspect, the invention provides cells transformed
with a nucleic acid including (a) a first 9 base pair DNA sequence
which can be bound by an I-CreI-derived meganuclease monomer or by
a first domain from a single-chain I-CreI-derived meganuclease; (b)
a four base pair DNA sequence selected from Table 2; and (c) a
second 9 base pair DNA sequence which can be bound by an
I-CreI-derived meganuclease monomer or by a second domain from a
single-chain I-CreI-derived meganuclease; wherein the second 9 base
pair DNA sequence is in the reverse orientation relative to the
first.
[0096] In another aspect, the invention provides a cell containing
an exogenous nucleic acid sequence integrated into its genome,
including, in order: (a) a first exogenous 9 base pair DNA sequence
which can be bound by an I-CreI-derived meganuclease monomer or by
a first domain from a single-chain I-CreI-derived meganuclease; (b)
an exogenous four base pair DNA sequence selected from Table 2; and
(c) a second exogenous 9 base pair DNA sequence which can be bound
by an I-CreI-derived meganuclease monomer or by a second domain
from a single-chain I-CreI-derived meganuclease; wherein the second
9 base pair DNA sequence is in the reverse orientation relative to
the first.
[0097] In another aspect, the invention provides a cell containing
an exogenous nucleic acid sequence integrated into its genome,
including, in order: (a) a first exogenous 9 base pair DNA sequence
which can be bound by an I-CreI-derived meganuclease monomer or by
a first domain from a single-chain I-CreI-derived meganuclease; (b)
an exogenous two base pair DNA sequence, wherein the two base pairs
correspond to bases N.sub.+1 and N.sub.+2 of a four base pair DNA
sequence selected from Table 2; (c) an exogenous DNA sequence
comprising a coding sequence which is expressed in the cell; (d) an
exogenous two base pair DNA sequence, wherein the two base pairs
correspond to bases N.sub.+3 and N.sub.+4 of a four base pair DNA
sequence selected from Table 2; and (e) a second exogenous 9 base
pair DNA sequence which can be bound by the I-CreI-derived
meganuclease monomer or by a second domain from the single-chain
I-CreI-derived meganuclease; wherein the second 9 base pair DNA
sequence is in the reverse orientation relative to the first.
[0098] In some embodiments, the nucleic acid is a plasmid. In other
embodiments, the nucleic acid is an artificial chromosome. In other
embodiments, the nucleic acid is integrated into the genomic DNA of
the cell. In other embodiments, the nucleic acid is a viral nucleic
acid.
[0099] In some embodiments, the cell is selected from the group a
human cell, a non-human animal cell, a plant cell, a bacterial
cell, and a fungal cell.
[0100] In some of the foregoing embodiments, the four base pair DNA
sequence is selected from Table 3. In other embodiments, the four
base pair DNA sequence is selected from Table 4.
[0101] In some embodiments, the I-CreI meganuclease is specifically
designed for use with the chosen recognition site in the methods of
the invention.
2.4 Methods of Conducting a Custom-Designed, I-CreI-Derived
Meganuclease Business
[0102] A meganuclease business can be conducted based on
I-CreI-derived meganucleases. For example, such business can
operate as following. The business received a DNA sequence into
which a double-strand break is to be introduced by a
rationally-designed I CreI-derived meganuclease. The business
identifies in the DNA sequence at least one recognition site for a
rationally-designed I CreI-derived meganuclease with altered
specificity relative to I-CreI, wherein the recognition site is not
cleaved by a naturally-occurring I-CreI, wherein the recognition
site has a four base pair central sequence selected from the group
consisting of TTGT, TTAT, TCTT, TCGT, TCAT, GTTT, GTCT, GGAT, GAGT,
GAAT, ATGT, TTTC, TTCC, TGAC, TAAC, GTTC, ATAT, TCGA, TTAA, GGGC,
ACGC, CCGC, CTGC, ACAA, ATAA, AAGA, ACGA, ATGA, AAAC, AGAC, ATCC,
ACTC, ATTC, ACAT, GAAA, GGAA, GTCA, GTTA, GAAC, ATAT, TCGA, TTAA,
GCCC, GCGT, GCGG and GCAG. The business then provides a
rationally-designed meganuclease that cleaves the recognition site
in the DNA.
[0103] Optionally, the business rationally-designs an
I-CreI-derived meganuclease that cleaves the recognition site in
the DNA. Optionally, the business produces the rationally-designed
I-CreI-derived meganuclease.
2.5 Specifically Excluded Center Sequences.
[0104] The center sequences GTAC, ACAC, and GTGA have previously
been shown to be effective center sequences for in vitro and in
vivo applications. These center sequences are specifically excluded
from some aspects of the present invention. In addition, the center
sequences TTGA and GAAA have previously been shown to be poor
center sequences for in vivo applications (Arnould, et al. (2007).
J. Mol. Biol. 371: 49-65).
EXAMPLES
[0105] This invention is further illustrated by the following
examples, which should not be construed as limiting. Those skilled
in the art will recognize, or be able to ascertain, using no more
than routine experimentation, numerous equivalents to the specific
substances and procedures described herein. Such equivalents are
intended to be encompassed in the scope of the claims that follow
the examples below. Examples 1 and 2 refer to engineered
meganucleases cleaving optimized meganuclease recognition sites in
vivo in a model plant system. Example 3 refers to an engineered
meganuclease cleaving optimized meganuclease recognition sites in
vitro.
Example 1
Cleavage of an Optimized Meganuclease Recognition Site by a
Rationally-Designed, I-CreI-Derived Meganuclease Homodimer in
vivo
[0106] An engineered meganuclease called BRP2 (SEQ ID NO: 8) was
produced using the method disclosed in WO 2007/047859. This
meganuclease is derived from I-CreI and was engineered to recognize
DNA sites that are not recognized by wild-type I-CreI (e.g., BRP2
recognition sequences include SEQ ID NO: 9 and SEQ ID NO: 10, or
SEQ ID NO: 11 and SEQ ID NO: 12). To facilitate nuclear
localization of the engineered meganuclease, an SV40 nuclear
localization signal (NLS, SEQ ID NO: 13) was added to the
N-terminus of the protein. Conventional Agrobacterium-mediated
transformation procedures were used to transform Arabidopsis
thaliana with a T-DNA containing a codon-optimized BRP2 coding
sequence (SEQ ID NO: 14). Expression of BRP2 meganuclease was under
the control of a Hsp70 promoter and a NOS terminator. A pair of
BRP2 recognition sequences were housed on the same T-DNA separated
by 7 base pairs containing a PstI restriction enzyme site (FIG.
3a). BRP2 cutting of the pair of BRP2 recognition sequences in this
construct was expected to excise the region between the recognition
sequences and thereby remove the PstI restriction site (FIG. 3b).
Two such T-DNA constructs were produced which varied the center
sequence of the meganuclease recognition sequences flanking the
PstI restriction enzyme site (FIG. 3c). In the first construct (the
"GTAC construct"), the meganuclease recognition sites had the
center sequence GTAC (a preferred in vivo center sequence, Table 5,
sequence 9; SEQ ID NO: 9 and SEQ ID NO 10). The second construct
(the "TAGA construct") had the center sequence TAGA (a
non-preferred center sequence, opposite strand sequence to Table 2,
sequence 77; SEQ ID NO: 11 and SEQ ID NO 12).
[0107] Stably transformed Arabidopsis plants carrying each
construct were produced by selection for a kanamycin resistance
marker housed on the T-DNA. Genomic DNA was then isolated from the
transformed plants (by leaf punch) before and after heat-shock to
induce BRP2 meganuclease expression. Genomic DNA samples were added
to PCR reactions using primers to amplify the region of the T-DNA
housing the meganuclease recognition sequences. PCR products were
then digested with PstI and visualized by gel electrophoresis (FIG.
3d). Results are summarized in Table 6. Any PCR sample in which a
significant percentage (>25%) of product was found to be
resistant to PstI was considered to be indicative of in vivo
meganuclease cleavage in that particular plant and was scored as
"cut" in Table 6. It was found that, prior to heat-shock, the vast
majority of PCR samples from plants carrying either construct
retained the PstI site. After heat-shock, however, a large
percentage of samples taken from plants transformed with the GTAC
construct, but not the TAGA construct, had lost the PstI site. PCR
products from the GTAC construct-transformed plants lacking a PstI
site were cloned into a pUC-19 plasmid and sequenced. 100% of
sequenced clones had a precise deletion of the region between the
two BRP2 cut sites (as diagrammed in FIG. 3b). These results
indicate that an engineered meganuclease is able to cleave a
meganuclease recognition site in vivo provided it has an optimized
center sequence.
TABLE-US-00006 TABLE 6 In vivo cleavage of optimized meganuclease
recognition sequences by an engineered meganuclease homodimer.
Before heat-shock After heat-shock Construct Cut Uncut Cut Uncut
GTAC 0 4 3 1 TAGA 0 22 0 22
Example 2
Cleavage of an Optimized Meganuclease Recognition Site by a
Rationally-Designed, I-CreI-Derived Meganuclease Single-Chain
Heterodimer in vivo
[0108] The engineered meganuclease BRP12-SC (SEQ ID NO: 15) was
produced in accordance with WO 2007/047859, except that this
meganuclease is a single-chain heterodimer. As discussed in WO
2007/047859, wild-type I-CreI binds to and cleaves DNA as a
homodimer. As a consequence, the natural recognition sequence for
I-CreI is pseudo-palindromic. The BRP12-SC recognition sequences,
however, are non-palindromic (e.g., SEQ ID NO: 16 and SEQ ID NO:
17, or SEQ ID NO: 18 and SEQ ID NO: 19). This necessitates the use
of an engineered meganuclease heterodimer comprising a pair of
subunits each of which recognizes one half-site within the
full-length recognition sequence. In the case of BRP12-SC, the two
engineered meganuclease monomers are physically linked to one
another using an amino acid linker to produce a single-chain
heterodimer. This linker comprises amino acids 166-204 (SEQ ID NO:
20) of BRP12-SC. The linker sequence joins an N-terminal
meganuclease subunit terminated at L165 (corresponding to L155 of
wild-type I-CreI) with a C-terminal meganuclease subunit starting
at K204 (corresponding to K7 of wild-type I-CreI). The benefits of
physically linking the two meganuclease monomers using this novel
linker is twofold: First, it ensures that the meganuclease monomers
can only associate with one another (heterodimerize) to cut the
non-palindromic BRP12-SC recognition sequence rather than also
forming homodimers which can recognize palindromic or
pseudopalindromic DNA sites that differ from the BRP12-SC
recognition sequence. Second, the physical linking of meganuclease
monomers obviates the need to express two monomers simultaneously
in the same cell to obtain the desired heterodimer. This
significantly simplifies vector construction in that it only
requires a single gene expression cassette. As was the case with
the BRP2 meganuclease discussed in Example 1, the BRP12-SC
meganuclease has an SV40 nuclear localization signal (SEQ ID NO:
13) at its N-terminus.
[0109] Conventional Agrobacterium-mediated transformation
procedures were used to transform Arabidopsis thaliana with a T-DNA
containing a codon-optimized BRP12-SC coding sequence (SEQ ID NO:
21). Expression of BRP12-SC meganuclease was under the control of a
Hsp70 promoter and a NOS terminator. A pair of BRP12-SC recognition
sequences were housed on the same T-DNA separated by 7 base pairs
containing a PstI restriction enzyme site (FIG. 4a). BRP12-SC
cutting of the pair of BRP12-SC recognition sequences in this
construct was expected to excise the region between the recognition
sequences and thereby remove the PstI restriction site (FIG. 4b).
Two such T-DNA constructs were produced which varied only in the
center sequences of the meganuclease recognition sequences flanking
the PstI restriction enzyme site (FIG. 4c). In the first construct
(the "GTAC construct"), the meganuclease recognition sites had the
center sequence GTAC (a preferred in vivo center sequence, Table 5,
sequence 9; SEQ ID NO: 16 and SEQ ID NO 17). The second construct
(the "TAGA construct") had the center sequence TAGA (a
non-preferred center sequence, opposite strand sequence to Table 2,
sequence 77; SEQ ID NO: 18 and SEQ ID NO 19).
[0110] Stably transformed Arabidopsis plants carrying each
construct were produced by selection for a kanamycin resistance
marker housed on the T-DNA. Genomic DNA was then isolated from the
transformed plants (by leaf punch) before and after heat-shock to
induce BRP12-SC meganuclease expression. Genomic DNA samples were
added to PCR reactions using primers to amplify the region of the
T-DNA housing the meganuclease recognition sequences. PCR products
were then digested with PstI and visualized by gel electrophoresis.
The results of this analysis are presented in Table 7. Any PCR
sample in which a significant percentage (>25%) of product was
found to be resistant to PstI was considered to be indicative of in
vivo meganuclease cleavage and was scored as "cut" in Table 7. It
was found that, prior to heat-shock, the vast majority of PCR
samples from plants carrying either construct retained the PstI
site. After heat-shock, however, a large percentage of samples
taken from plants transformed with the GTAC construct, but not the
TAGA construct, had lost the PstI site. PCR products from the GTAC
construct-transformed plants lacking a PstI site were cloned into a
pUC-19 plasmid and sequenced. 100% of sequenced clones had a
precise deletion of the region between the two BRP12-SC cut sites
(as diagrammed in FIG. 4b). These results indicate that an
engineered single chain meganuclease is able to cleave a
meganuclease recognition site in vivo provided it has an optimized
center sequence.
TABLE-US-00007 TABLE 7 In vivo cleavage of optimized meganuclease
recognition sequences by an engineered meganuclease homodimer.
Before heat-shock After heat-shock Construct Cut Uncut Cut Uncut
GTAC 0 23 8 15 TAGA 0 59 1 58
Example 3
Cleavage of an Optimized Meganuclease Recognition Site by a
Rationally-Designed, I-CreI-Derived Meganuclease Homodimer In
Vitro
[0111] The BRP2 meganuclease described in Example 1 (SEQ ID NO: 8)
was expressed in E. coli and purified as in Example 1 of WO
2007/047859. The purified meganuclease was then added at varying
concentrations to reactions containing plasmids harboring BRP2
recognition sequences with either a GTAC or TAGA center sequence
(0.25 picomoles of plasmid substrate in 25 microliters of SA
buffer: 25 mM Tris-HCL, pH 8.0, 100 mM NaCl, 5 mM MgCl.sub.2, 5 mM
EDTA). Reactions were incubated at 37.degree. C. for 2 hours and
were then visualized by gel electrophoresis and the percentage of
each plasmid substrate cleaved by the meganuclease was plotted as a
function of meganuclease concentration (FIG. 5). It was found that
the plasmid substrate with the TAGA center sequence was cleaved by
the meganuclease in vitro, but that cleavage of this substrate
required a far higher concentration of BRP2 meganuclease than did
cleavage of the GTAC substrate.
Sequence CWU 1
1
231163PRTChlamydomonas reinhardtii 1Met Asn Thr Lys Tyr Asn Lys Glu
Phe Leu Leu Tyr Leu Ala Gly Phe1 5 10 15Val Asp Gly Asp Gly Ser Ile
Ile Ala Gln Ile Lys Pro Asn Gln Ser 20 25 30Tyr Lys Phe Lys His Gln
Leu Ser Leu Ala Phe Gln Val Thr Gln Lys 35 40 45Thr Gln Arg Arg Trp
Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val 50 55 60Gly Tyr Val Arg
Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu65 70 75 80Ile Lys
Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys 85 90 95Leu
Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Trp Arg Leu 100 105
110Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys
Thr Thr 130 135 140Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser
Glu Lys Lys Lys145 150 155 160Ser Ser Pro222DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 2gaaactgtct cacgacgttt tg 22322DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 3caaaacgtcg tgagacagtt tc 224163PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
4Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe1 5
10 15Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Asp Pro Arg Gln
Asn 20 25 30Tyr Lys Phe Lys His Glu Leu Arg Leu Arg Phe Gln Val Thr
Gln Lys 35 40 45Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu
Ile Gly Val 50 55 60Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr
Ile Leu Ser Glu65 70 75 80Ile Lys Pro Leu His Asn Phe Leu Thr Gln
Leu Gln Pro Phe Leu Lys 85 90 95Leu Lys Gln Lys Gln Ala Asn Leu Val
Leu Lys Ile Ile Glu Gln Leu 100 105 110Pro Ser Ala Lys Glu Ser Pro
Asp Lys Phe Leu Glu Val Cys Thr Trp 115 120 125Val Asp Gln Ile Ala
Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr 130 135 140Ser Glu Thr
Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys145 150 155
160Ser Ser Pro522DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 5aacggtgtcg tgagacaccg tt
22622DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 6aacggtgtct cacgacaccg tt
2279DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 7aacggtgtc 98173PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
8Met Gly Pro Lys Lys Lys Arg Lys Val Ile Met Asn Thr Lys Tyr Asn1 5
10 15Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe Val Asp Gly Asp Gly
Ser 20 25 30Ile Ile Ala Ser Ile Arg Pro Arg Gln Ser Cys Lys Phe Lys
His Glu 35 40 45Leu Glu Leu Arg Phe Gln Val Thr Gln Lys Thr Gln Arg
Arg Trp Phe 50 55 60Leu Asp Lys Leu Val Asp Glu Ile Gly Val Gly Tyr
Val Arg Asp Arg65 70 75 80Gly Ser Val Ser Asp Tyr Arg Leu Ser Gln
Ile Lys Pro Leu His Asn 85 90 95Phe Leu Thr Gln Leu Gln Pro Phe Leu
Lys Leu Lys Gln Lys Gln Ala 100 105 110Asn Leu Val Leu Lys Ile Ile
Glu Gln Leu Pro Ser Ala Lys Glu Ser 115 120 125Pro Asp Lys Phe Leu
Glu Val Cys Thr Trp Val Asp Gln Ile Ala Ala 130 135 140Leu Asn Asp
Ser Lys Thr Arg Lys Thr Thr Ser Glu Thr Val Arg Ala145 150 155
160Val Leu Asp Ser Leu Ser Glu Lys Lys Lys Ser Ser Pro 165
170922DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 9ctccgggtcg tacgacccgg ag
221022DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 10ctccgggtcg tacgacccgg ag
221122DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 11ctccgggtct agagacccgg ag
221222DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 12ctccgggtct ctagacccgg ag
22139PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Met Ala Pro Lys Lys Lys Arg Lys Val1
514522DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 14atgggcccga agaagaagcg caaggtcatc
atgaacacca agtacaacaa ggagttcctg 60ctctacctgg cgggcttcgt ggacggggac
ggctccatca tcgcctccat ccgcccgcgt 120cagtcctgca agttcaagca
tgagctggaa ctccggttcc aggtcacgca gaagacacag 180cgccgttggt
tcctcgacaa gctggtggac gagatcgggg tgggctacgt gcgcgaccgc
240ggcagcgtct ccgactaccg cctgagccag atcaagcctc tgcacaactt
cctgacccag 300ctccagccct tcctgaagct caagcagaag caggccaacc
tcgtgctgaa gatcatcgag 360cagctgccct ccgccaagga atccccggac
aagttcctgg aggtgtgcac ctgggtggac 420cagatcgccg ctctgaacga
ctccaagacc cgcaagacca cttccgagac cgtccgcgcc 480gtgctggaca
gtctctccga gaagaagaag tcgtccccct ag 52215360PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
15Met Gly Pro Lys Lys Lys Arg Lys Val Ile Met Asn Thr Lys Tyr Asn1
5 10 15Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe Val Asp Gly Asp Gly
Ser 20 25 30Ile Lys Ala Gln Ile Arg Pro Arg Gln Ser Arg Lys Phe Lys
His Glu 35 40 45Leu Glu Leu Thr Phe Gln Val Thr Gln Lys Thr Gln Arg
Arg Trp Phe 50 55 60Leu Asp Lys Leu Val Asp Glu Ile Gly Val Gly Lys
Val Tyr Asp Arg65 70 75 80Gly Ser Val Ser Asp Tyr Glu Leu Ser Gln
Ile Lys Pro Leu His Asn 85 90 95Phe Leu Thr Gln Leu Gln Pro Phe Leu
Lys Leu Lys Gln Lys Gln Ala 100 105 110Asn Leu Val Leu Lys Ile Ile
Glu Gln Leu Pro Ser Ala Lys Glu Ser 115 120 125Pro Asp Lys Phe Leu
Glu Val Cys Thr Trp Val Asp Gln Ile Ala Ala 130 135 140Leu Asn Asp
Ser Lys Thr Arg Lys Thr Thr Ser Glu Thr Val Arg Ala145 150 155
160Val Leu Asp Ser Leu Pro Gly Ser Val Gly Gly Leu Ser Pro Ser Gln
165 170 175Ala Ser Ser Ala Ala Ser Ser Ala Ser Ser Ser Pro Gly Ser
Gly Ile 180 185 190Ser Glu Ala Leu Arg Ala Gly Ala Thr Lys Ser Lys
Glu Phe Leu Leu 195 200 205Tyr Leu Ala Gly Phe Val Asp Gly Asp Gly
Ser Ile Ile Ala Ser Ile 210 215 220Arg Pro Arg Gln Ser Cys Lys Phe
Lys His Glu Leu Glu Leu Arg Phe225 230 235 240Gln Val Thr Gln Lys
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val 245 250 255Asp Glu Ile
Gly Val Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp 260 265 270Tyr
Arg Leu Ser Gln Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu 275 280
285Gln Pro Phe Leu Lys Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys
290 295 300Ile Ile Glu Gln Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys
Phe Leu305 310 315 320Glu Val Cys Thr Trp Val Asp Gln Ile Ala Ala
Leu Asn Asp Ser Lys 325 330 335Thr Arg Lys Thr Thr Ser Glu Thr Val
Arg Ala Val Leu Asp Ser Leu 340 345 350Ser Glu Lys Lys Lys Ser Ser
Pro 355 3601622DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 16tgcctcctcg tacgacccgg ag
221722DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 17ctccgggtcg tacgaggagg ca
221822DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 18tgcctcctct agagacccgg ag
221922DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 19ctccgggtct ctagaggagg ca
222038PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 20Pro Gly Ser Val Gly Gly Leu Ser Pro Ser Gln
Ala Ser Ser Ala Ala1 5 10 15Ser Ser Ala Ser Ser Ser Pro Gly Ser Gly
Ile Ser Glu Ala Leu Arg 20 25 30Ala Gly Ala Thr Lys Ser
35211083DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 21atgggcccga agaagaagcg caaggtcatc
atgaacacca agtacaacaa ggagttcctg 60ctctacctgg ccggcttcgt ggacggcgac
ggctccatca aggcgcagat ccgtccgcgg 120cagagccgga agttcaagca
cgagctcgag ctgaccttcc aggtgaccca gaagacgcag 180aggcgctggt
tcctcgacaa gctggtggac gagatcgggg tgggcaaggt ctacgaccgc
240gggtcggtgt ccgactacga gctctcccag atcaagcccc tgcacaactt
cctcacccag 300ctccagccgt tcctgaagct caagcagaag caggccaacc
tcgtgctgaa gatcatcgag 360cagctgccct ccgccaagga atccccggac
aagttcctgg aggtgtgcac gtgggtggac 420cagatcgcgg ccctcaacga
cagcaagacc cgcaagacga cctcggagac ggtgcgggcg 480gtcctggact
ccctcccagg atccgtggga ggtctatcgc catctcaggc atccagcgcc
540gcatcctcgg cttcctcaag cccgggttca gggatctccg aagcactcag
agctggagca 600actaagtcca aggaattcct gctctacctg gcgggcttcg
tggacgggga cggctccatc 660atcgcctcca tccgcccgcg tcagtcctgc
aagttcaagc atgagctgga actccggttc 720caggtcacgc agaagacaca
gcgccgttgg ttcctcgaca agctggtgga cgagatcggg 780gtgggctacg
tgcgcgaccg cggcagcgtc tccgactacc gcctgagcca gatcaagcct
840ctgcacaact tcctgaccca gctccagccc ttcctgaagc tcaagcagaa
gcaggccaac 900ctcgtgctga agatcatcga gcagctgccc tccgccaagg
aatccccgga caagttcctg 960gaggtgtgca cctgggtgga ccagatcgcc
gctctgaacg actccaagac ccgcaagacc 1020acttccgaga ccgtccgcgc
cgtgctggac agtctctccg agaagaagaa gtcgtccccc 1080tag
10832222DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 22aacggtgtcn nnngacaccg tt
222322DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 23aacggtgtcn nnngacaccg tt 22
* * * * *