JPWO2020264016A5

JPWO2020264016A5 -

Info

Publication number: JPWO2020264016A5
Application number: JP2021576666A
Authority: JP
Publication date: 2023-07-04

Claims

A method of increasing homologous recombination repair (HDR)-mediated genomic modification of a targeted editing site in a eukaryotic genome, comprising:
Providing a eukaryotic cell with a genome-editing molecule and an HDR-enhancing agent, wherein the genome- editing molecule comprises (i) at least one sequence-specific endonuclease or sequence that cleaves a DNA sequence in the target editing site; (ii) a donor template DNA molecule having homology to a target editing site; and an HDR facilitator comprising a single-stranded DNA annealing protein ( SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB);
A method, wherein the genome -editing molecule and the HDR-enhancing agent result in targeted editing site modification of a eukaryotic cell genome with a donor template polynucleotide by HDR at an increased frequency compared to a control.

A sequence- specific endonuclease is
(a) comprising an RNA-guided nuclease or a polynucleotide encoding an RNA-guided nuclease and a guide RNA or a polynucleotide encoding the guide RNA;
(b) zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TAL effector nucleases), Argonautes, meganucleases, or engineered meganucleases;
(c) comprising an RNA-guided nuclease, wherein the target editing site comprises a PAM sequence and a sequence complementary to the guide RNA and located directly adjacent to the protospacer adjacent motif (PAM) sequence; and/or
(d) providing a 5' overhang to the target editing site after cleavage;
The method of claim 1.

4. The genome editing molecule comprises one or more sequence-specific endonucleases or a sequence-specific endonuclease and a guide RNA that cleave a single DNA strand at two separate DNA sequences at the target editing site. 3. The method according to 1 or 2 .

(a) the sequence- specific endonuclease comprises at least one Cas9 nickase, Cas12a nickase, Cas12i, zinc finger nickase, TALE nickase, or a combination thereof;
(b) the sequence-specific endonuclease comprises Cas9 and/or Cas12a and the guide RNA molecule has at least one base mismatch with the DNA sequence of the target editing site; and/or
(c) the exonuclease is at least 70%, 75%, 80% with T7 phage exonuclease, E. coli exonuclease III, a related protein with comparable exonuclease activity, or SEQ ID NO: 143 or 144; comprising proteins with 85%, 90%, 95%, or 99% sequence identity;
4. The method of claim 3 .

a donor template DNA molecule
(a) provided on a circular DNA vector, a geminivirus replicon, or as a linear DNA fragment; and/or
(b) flanked by copies of the endonuclease recognition sequence;
The method according to any one of claims 1-4 .

S SAP is
(a) provide for DNA strand exchange and base pairing of complementary DNA strands of homologous DNA molecules; and/or
(b) a RecT/Redβ family protein, an ERF family protein, or a RAD52 family protein,
(i) the RecT/Redβ family protein is at least the Rac bacterial prophage RecT protein, the bacteriophage λbeta protein, the bacteriophage SPP1 35 protein, a related protein with comparable SSAP activity, or SEQ ID NO: 1, 2, or 3; including proteins with 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity;
(ii) the ERF family protein is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% the bacteriophage P22 ERF protein, a functionally related protein, or SEQ ID NO:4 contains a protein having the sequence identity of
(iii) the RAD52 family protein is Saccharomyces cerevisiae Rad52 protein, Schizosaccharomyces pombe Rad22 protein, Kluyveromyces lactis Rad52 protein , functionally related proteins or a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 5, 6, or 7;
The method according to any one of claims 1-5.

A system for increasing homologous recombination repair (HDR)-mediated genomic modification of targeted editing sites in a eukaryotic genome, comprising:
(a) a eukaryotic cell;
(b) a single-stranded DNA annealing protein (SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB); and (c) at least one sequence-specific endonuclease that cleaves a DNA sequence in the target editing site or at least one polynucleotide encoding a sequence -specific endonuclease, and the target editing site comprising one or more genome editing molecules comprising a donor template DNA molecule having homology to
A system wherein eukaryotic cells are associated with, contacted with and/or contain an effective amount of an HDR facilitating agent and one or more genome editing molecules.

Genome -editing molecules
(a) comprising an RNA-guided nuclease or a polynucleotide encoding the RNA-guided nuclease and a guide RNA or a polynucleotide encoding the guide RNA;
(b) zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TAL effector nucleases), Argonautes, meganucleases, or engineered meganucleases;
(c) one or more sequence-specific endonucleases or a sequence-specific endonuclease and a guide RNA that cleave a single DNA strand at two separate DNA sequences at the target editing site, wherein the sequence-specific endonuclease is ,
(i) comprises at least one Cas9 nickase, Cas12a nickase, Cas12i, zinc finger nickase, TALE nickase, or combinations thereof; and/or
(ii) comprising Cas9 and/or Cas12a, wherein the guide RNA molecule has at least one base mismatch with the DNA sequence of the target editing site;
(d) comprising an RNA-guided nuclease, wherein the target editing site comprises a PAM sequence and a sequence complementary to the guide RNA and located directly adjacent to the PAM sequence; and/or
(e) comprising at least one sequence-specific endonuclease, the sequence-specific endonuclease providing a 5' overhang to the target editing site after cleavage;
A system according to claim 7 .

8. The genome editing molecule and the HDR facilitator result in target editing site modification of the eukaryotic genome with the donor template polynucleotide by HDR at least 2-fold increased frequency compared to control. Or the system according to 8 .

S SAP is
(a) providing DNA strand exchange and base pairing of complementary DNA strands of homologous DNA molecules;
(b) a RecT/Redβ family protein, an ERF family protein, or a RAD52 family protein,
(i) the RecT/Redβ family protein comprises the Rac bacterial prophage RecT protein, the bacteriophage λbeta protein, the bacteriophage SPP1 35 protein, or related proteins with comparable SSAP activity;
(ii) the RecT/Redβ family protein comprises a bacteriophage lambda beta protein, a bacteriophage SPP1 35 protein, a Rac bacterial prophage RecT protein, or a related protein with comparable SSAP activity;
(iii) RecT/Redβ family proteins include proteins having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 1, 2, or 3 ,
(iv) the ERF family protein is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% a bacteriophage P22 ERF protein, a functionally related protein, or SEQ ID NO:4 and/or
(v) the RAD52 family protein is Saccharomyces cerevisiae Rad52 protein, Schizosaccharomyces pombe Rad22 protein, Kluyveromyces lactis Rad52 protein, function related proteins or a protein having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 5, 6, or 7;
A system according to any one of claims 7-9 .

exonuclease is
(a) having preferred substrates comprising linear dsDNA molecules;
(b) having a preferred substrate comprising a linear dsDNA molecule containing a phosphorylated 5'end;
(c) blunt-ended dsDNA substrates, dsDNA substrates with internal breaks in one strand, dsDNA substrates with 5′ overhangs, and/or with 3′ overhangs, which have 5′→3′ exonuclease activity; capable of recognizing a dsDNA substrate,
(d) blunt-ended dsDNA substrates, dsDNA substrates with internal breaks in one strand, dsDNA substrates with 5′ overhangs, and/or with 3′ overhangs, which have 3′→5′ exonuclease activity; capable of recognizing dsDNA substrates and/or
(e) Bacteriophage lambda exoprotein, Rac prophage RecE exonuclease, Artemis protein, Apollo protein, DNA2 exonuclease, Exo1 exonuclease, Herpes virus SOX protein, UL12 exonuclease, enterobacterial exonuclease VIII, T7 phage exonuclease , E. coli exonuclease III, mammalian Trex2 exonuclease, related proteins with equivalent exonuclease activity, or proteins having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with 144 or 145;
A method according to any one of claims 1-6, or a system according to any one of claims 7-10 .

single -stranded DNA binding protein (SSB)
(a) obtained from the same host organism as the S SAP,
(b) is bacterial SSB;
(c) is Enterobacteriaceae sp. SSB;
(d) Escherichia sp., Shigella sp., Enterobacter sp., Klebsiella sp., Serratia sp., Pantoea sp. or Yersinia sp., and/or
(e) proteins having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs: 31 or 34-132;
A method according to any one of claims 1-6 and 11, or a system according to any one of claims 7-11 .

(a) said HDR frequency is increased at least 2-fold compared to a control system in which control eukaryotic cells are provided with a genome- editing molecule but not exposed to at least one HDR-enhancing agent; and/or
(b) the frequency of non-homologous end joining (NHEJ) is maintained compared to a control method or system in which control eukaryotic cells are provided with a genome-editing molecule but not exposed to at least one of said HDR-enhancing agents; or is at least reduced by a factor of two,
A method according to any one of claims 1-6 and 11-12, or a system according to any one of claims 7-12 .

S SAPs, exonucleases , and/or single -stranded DNA binding proteins (SSBs)
(a) further comprising an operably linked nuclear localization signal (NLS) and/or a cell penetrating peptide (CPP), and/or
(b) provided to the cell as a polyprotein comprising protease recognition sites or self-processing protein sequences inserted between SSAPs, exonucleases, and/or SSBs;
A method according to any one of claims 1-6 and 11-13, or a system according to any one of claims 7-13 .

The method of any one of claims 1-6 and 11-14, or the system of any one of claims 7-14 , wherein the eukaryotic cells are mammalian or plant cells.

the cells are plant cells,
(a) the plant cell is haploid, diploid, or polyploid;
(b) the plant cell is in a culture medium, in a plant, or in a plant tissue;
(c) the group in which the SSAP, exonuclease, and/or single-stranded DNA binding protein consists of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16 and/or further comprising an operably linked nuclear localization signal (NLS) selected from
(d) the system provides isolating and/or growing a plant cell, propagule obtained from the plant cell, or plant comprising the genomic modification; including modifications,
16. The system of claim 15 .

A eukaryotic cell comprising an HDR facilitating agent, a genome editing molecule and a eukaryotic cell or genome modification is provided in an array comprising a plurality of containers, compartments or locations, each container, compartment or location being associated with an HDR facilitating The method of any one of claims 1-6 and 11-15 , or any one of claims 7-16, comprising the agent, the genome editing molecule and the eukaryotic cell or eukaryotic cell comprising the genome modification. The system described in paragraph.

A method of genetically engineering a eukaryotic cell, comprising:
i) at least one sequence-specific endonuclease ;
ii) a donor template DNA molecule with homology to a eukaryotic target editing site ;
iii) single-stranded DNA annealing protein (SSAP) ,
iv) an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and
v) single-stranded DNA binding protein (SSB )
wherein the target editing site of the cell is modified by the donor template DNA molecule.

at least one sequence-specific endonuclease
(a) comprising an RNA-guided nuclease or a polynucleotide encoding an RNA-guided nuclease , wherein the method further provides the eukaryotic cell with the guide RNA or a polynucleotide encoding the guide RNA;
(b) a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TAL effector nuclease), an Argonaute, a meganuclease, or an engineered meganuclease; and/or
(c) a nickase,
19. The method of claim 18 .

The RNA -guided nuclease is an RNA-guided DNA endonuclease, a type II Cas nuclease, a Cas9 nuclease, a type V nuclease, a Cas12a nuclease, a Cas12b nuclease, a Cas12c nuclease, a CasY nuclease, a CasX nuclease, Cas12i, Cas14, or engineered The method of any one of claims 2-6, 11-15 and 17-19, or the system of any one of claims 8-17, comprising a nuclease.

21. Any one of claims 18-20, further comprising a guide RNA, wherein the sequence- specific endonuclease and the guide RNA cleave a single DNA strand at two separate DNA sequences at the target editing site. described method.

A sequence- specific endonuclease is
(a) the sequence-specific endonuclease comprises at least one Cas9 nickase, Cas12a nickase, zinc finger nickase, TALE nickase, or a combination thereof, wherein the sequence -specific endonuclease is specific for the endonuclease recognition sequence of the target editing site; and/or
(b) comprising Cas9 and/or Cas12a, wherein the guide RNA molecule has at least one base mismatch with the DNA sequence of the target editing site;
A method according to any one of claims 18-21 .

a donor template DNA molecule
(a) provided in a plasmid or geminivirus genome , and/or
(b) flanked by endonuclease recognition sequences;
A system according to any one of claims 7-17 or a method according to any one of claims 18-22 .

24. The method of any one of claims 18-23 , wherein the S SAP comprises a RecT/Redβ family protein , an ERF family protein , or a RAD52 family protein.

25. The method of claim 24 , wherein the RecT /Red[beta] family protein comprises the Rac bacterial prophage RecT protein, the bacteriophage [lambda]beta protein, the bacteriophage SPPl 35 protein, or related proteins with equivalent SSAP activity.

exonuclease is
(a) having preferred substrates for exonucleases comprising linear dsDNA molecules;
(b) having a preferred substrate comprising a linear dsDNA molecule containing a phosphorylated 5'end;
(c) blunt-ended dsDNA substrates, dsDNA substrates with internal breaks in one strand, dsDNA substrates with 5′ overhangs, and/or with 3′ overhangs, which have 5′→3′ exonuclease activity; capable of recognizing a dsDNA substrate,
(d) blunt-ended dsDNA substrates, dsDNA substrates with internal breaks in one strand, dsDNA substrates with 5′ overhangs, and/or with 3′ overhangs, which have 3′→5′ exonuclease activity; capable of recognizing a dsDNA substrate,
(e), bacteriophage lambda exoprotein, Rac prophage RecE exonuclease, artemis protein, apollo protein, DNA2 exonuclease, Exo1 exonuclease, herpes virus SOX protein, UL12 exonuclease, enterobacterial exonuclease VIII, T7 phage exo nuclease, E. coli exonuclease III, mammalian Trex2 exonuclease, related proteins with equivalent exonuclease activity, or SEQ ID NOs:8, 9, 136, 137, 138, 139, 140, 141, 142, 143 , 144, or 145, and/or
(f) T7 phage exonuclease, E. coli exonuclease III, related proteins with equivalent exonuclease activity, or at least 70%, 75%, 80%, 85%, 90 with SEQ ID NO: 143 or 144 %, 95%, or 99% sequence identity,
A method according to any one of claims 18-25 .

A method according to any one of claims 18-26, wherein the single-stranded DNA binding protein (SSB) and the SSAP are obtained from the same host organism.

A method according to any one of claims 18-27 , wherein the eukaryotic cell is a mammalian cell or a plant cell.

the cells are plant cells,
(a) the plant cell is haploid, diploid, or polyploid;
(b) the plant cell is in a culture medium, in a plant, or in a plant tissue; and/or
(c) the method further comprises isolating and/or growing the plant cell, propagule obtained from the plant cell, plant comprising the genomic modification, wherein the genome of the plant cell, propagule, or plant comprises the genomic modification; ,
29. The method of claim 28 .

i ) at least one sequence-specific endonuclease, ii) a donor template DNA molecule with homology to a eukaryotic target editing site, iii) a single-stranded DNA annealing protein (SSAP), iv) a double-stranded DNA substrate. into a single- stranded DNA product, and v) a single-stranded DNA binding protein (SSB),
(a) provided on one or more vectors,
(b) provided on a chromosome;
(c) introduced polypeptides, DNA, mRNA and/or provided by sexual mating; and/or
(d) provided by a progenitor cell comprising one or more of i)-v), wherein the progenitor cell does not comprise at least one of i)-v) and is not included in the progenitor cell i) at least one of -v) is subsequently provided to the progenitor cell by delivery of the polypeptide, DNA or mRNA to the progenitor cell and/or by sexual mating of the progenitor cell;
A method according to any one of claims 18-29 .

31. The method of claim 30 , wherein the vector is an Agrobacterium vector.

(a) further comprising detecting the modification ;
(b) the target editing site is in the protein coding sequence or in the promoter;
(c) the modification of the target editing site is an insertion, deletion, or substitution;
(d) the target editing site is a gene encoding an agronomically important trait or a gene involved in a mammalian disease;
(e) the method further comprises isolating or growing eukaryotic cells comprising the modified target editing site;
(f) homologous recombination repair (HDR) frequency is increased by at least 2-fold compared to the control system, wherein the control eukaryotic cell provides at least at least one sequence-specific endonuclease and a donor template DNA molecule; but not exposed to at least one of the group consisting of SSAP, exonuclease, and SSB;
(g) the frequency of non-homologous end joining (NHEJ) is maintained or reduced by at least 2-fold compared to a control method or system not exposed to at least one of the HDR-enhancing agents, wherein control eukaryotic cells are provided with at least one sequence-specific endonuclease and a donor template DNA molecule, but are not exposed to at least one of the group consisting of SSAP, exonuclease, and SSB;
A method according to any one of claims 18-31 .

33. The method of claim 32 , wherein detecting modifications comprises amplicon sequencing.

A method for producing a eukaryotic cell having a genetically modified target editing site, comprising:
(a) providing at least one sequence-specific endonuclease that cleaves at least one endonuclease recognition sequence DNA sequence in said target editing site or at least one polynucleotide encoding said at least one sequence-specific endonuclease; and (b) providing at least one donor molecule comprising at least one double-stranded DNA sequence, wherein (i) said DNA sequence comprises sequences flanking the target editing site and at least 50 nucleotides of has at least 90% homology over its length, and (ii) said donor sequence contains at least one modification relative to said target editing site; and (c) at least one homologous recombination repair ( HDR) enhancer, wherein the HDR enhancer comprises
(i) at least one single-stranded DNA annealing protein (SSAP); and (ii) at least one exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product; iii) at least one single-stranded DNA binding protein (SSB)
includes;
and whereby at least one sequence-specific endonuclease, at least one donor molecule, and at least one HDR facilitator introduce said modification into said target editing site of said eukaryotic cell; and (d) isolating or growing a eukaryotic cell containing a modification at said target editing site;
method including.

(a) the modification is selected from the group consisting of insertion of one or more nucleotides, deletion of one or more nucleotides, or substitution of one or more nucleotides; and/or
(b) a portion of the target editing site is deleted by using two sequence-specific cleavages at said target editing site and replaced with a sequence provided by a donor molecule;
35. The method of claim 34 .

(a) the donor molecule is flanked by endonuclease recognition sequences in the vector, and/or
(b) further comprising growing the eukaryotic cell comprising the modification;
36. A method according to claim 34 or 35 .

A method of making a genetically modified organism, comprising:
37. A method comprising the steps of (i) producing a genetically modified eukaryotic cell according to any one of claims 34-36 , and (ii) regenerating said cell into an organism.

38. The method of claim 37 , wherein the organism is selected from the group consisting of plants and non-human animals.

i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule with homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) a double-stranded DNA substrate. A composition comprising a nucleic acid encoding one or more of an exonuclease capable of at least partially converting to a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB).

40. The composition of claim 39, wherein the nucleic acids are in one or more vectors.

i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule with homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) a double-stranded DNA substrate. A vector comprising a nucleic acid encoding one or more of an exonuclease that can be at least partially converted into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB).

the vector is
(a) comprising a donor template DNA, a sequence- specific endonuclease, and a polynucleotide encoding a guide RNA;
(b) a single-stranded DNA annealing protein (SSAP), an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and a single-stranded DNA binding protein (SSB); including,
(c) i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule with homology to a eukaryotic target editing site, iii) a single-stranded DNA annealing protein (SSAP), iv) double-stranded an exonuclease capable of at least partially converting a DNA substrate into a single-stranded DNA product; and v) a nucleic acid encoding a single-stranded DNA binding protein (SSB); and/or
(d) a sequence-specific nuclease operably linked to an inducible promoter;
42. The vector of claim 41.

i) at least one sequence-specific endonuclease, ii) a donor template DNA molecule with homology to a target editing site in a eukaryotic cell, iii) a single-stranded DNA annealing protein (SSAP), iv) a double-stranded DNA substrate. an exonuclease capable of at least partially converting to a single-stranded DNA product; and v) a nucleic acid encoding a single-stranded DNA binding protein (SSB) and instructions for genetic manipulation of eukaryotic cells kit.

the kit is
(a) a first vector and a second vector, wherein i) the first vector comprises a nucleic acid comprising donor template DNA and a sequence -specific endonuclease, wherein the sequence- specific endonuclease comprises a polynucleotide encoding an RNA-guided nuclease and a polynucleotide encoding a guide RNA; and ii) a second vector comprises a single-stranded DNA annealing protein (SSAP) and a double-stranded DNA substrate. an exonuclease capable of at least partially converting into a single-stranded DNA product and a single-stranded DNA binding protein (SSB); and/or
(b) further comprising an agent for detection of genetically engineered cells;
44. The kit of claim 43 .

i) at least one sequence-specific endonuclease or a nucleic acid encoding at least one sequence-specific endonuclease , ii) a donor template DNA molecule with homology to a target editing site of a eukaryotic cell, iii) single-stranded DNA annealing. a protein (SSAP) or a nucleic acid encoding an SSAP , iv) an exonuclease or a nucleic acid encoding an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and v) a single A cell containing a strand DNA binding protein (SSB) or a nucleic acid encoding an SSB .

the cells
(a) is a plant or mammalian cell, and/or
(b) is a host cell;
46. The cell of claim 45 .

A genetically engineered cell produced by the method of any one of claims 1-6, 11-15, and 17-38 or the system of any one of claims 7-17 .

A progenitor eukaryotic cell or organism for genetic manipulation at a target editing site, comprising: i) at least one sequence-specific endonuclease; ii) a donor template molecule having homology to the target editing site of the eukaryotic cell; iii) a single-stranded DNA annealing protein (SSAP), iv) an exonuclease capable of at least partially converting a double-stranded DNA substrate into a single-stranded DNA product, and v) a single-stranded DNA binding protein (SSB). at least one of i) to v), wherein the cell does not include at least one of i) to v), wherein the progenitor cell or organism is not included in the cell or A progenitor eukaryotic cell or organism that, when provided to the organism, will be modified at the target editing site by the donor template molecule.

(a) the donor template is a double-stranded DNA molecule;
(b) the cell is a germline cell;
(c) the cell is a progenitor plant cell and at least one of i) to v) contained in the progenitor plant cell or plant is provided by transformation;
(d) a second plant wherein the organism is a plant and at least one of i) to v) not included in the progenitor plant includes at least one of i) to v) not included in the progenitor plant supplied by sexual interbreeding with
(e) the eukaryotic progenitor cell is an animal cell and at least one of i) to v) not contained in progenitor cells is provided by transfection, and/or
(f) the precursor is a non-human animal, and at least one of i) to v) not included in non-human animals includes at least one of i) to v) not included in non-human animals supplied by sexual crosses with non-human animals, including
49. A progenitor eukaryotic cell or organism according to claim 48 .