HK40111540A - Methods for large-size chromosomal transfer and modified chromosomes and organisms using same - Google Patents

Description

Large-size chromosome transfer methods and modified chromosomes and organisms produced using these methods.

By referencing and incorporating into the sequence list

This application contains a sequence list which has been submitted in ASCII format via the EFS website and is incorporated herein by reference in its entirety.

Background Technology

Manipulation of large segments of genes or chromosomes is a powerful tool for basic and translational research, as well as for therapies. Human genes range in size from hundreds of base pairs to at least 2,300 kilobases (KB), and human chromosomes range in size from 38 megabase pairs (MB) to nearly 250 MB. Therefore, efficient study of large genes, regions spanning multiple genes, and portions of chromosomes requires manipulation of large sequence fragments. However, large-fragment manipulation remains one of the most significant challenges in the field of gene editing. This disclosure provides methods for manipulating large sequences.

Summary of the Invention

This disclosure provides a method for generating engineered chromosomes, comprising: (a) providing cells comprising a target chromosome containing a target sequence and a template chromosome containing a template sequence; (b) contacting the cells with (i) a first nucleic acid molecule and (ii) a second nucleic acid molecule, the first nucleic acid molecule comprising, from 5' to 3', a 5' homologous arm, at least one first marker, and a 3' homologous arm, the 5' homologous arm containing a nucleotide sequence upstream of the 5' end of the target sequence, and the 3' homologous arm containing a nucleotide sequence upstream of the 5' end of the template sequence; the second nucleic acid molecule comprising, from 5' to 3', a 5' homologous arm, at least one second marker, and a 3' homologous arm, the 5' homologous arm containing a nucleotide sequence downstream of the 3' end of the template sequence, and the 3' homologous arm containing a nucleotide sequence downstream of the 3' end of the target sequence; (c) generating double-strand breaks at or on either side of the target sequence and at the 5' and 3' ends of the template sequence, thereby inserting the template sequence and the first and second markers into the target chromosome; and (d) selecting one or more cells expressing the first and second markers.

In some implementations, after inserting the template sequence, the first marker is located at the 5' end of the template sequence, and the second marker is located at the 3' end of the template sequence.

In some embodiments, the lengths of the 5' and 3' homologous arms of the first and second nucleic acid molecules are between about 20 and 2,000 base pairs (bp), between about 50 bp and 1,500 bp, between about 100 bp and 1,400 bp, between about 150 bp and 1,300 bp, between about 200 bp and 1,200 bp, between about 300 bp and 1,100 bp, between about 400 bp and 1,000 bp, or between about 500 bp and 900 bp, or between about 600 bp and 800 bp. In some embodiments, the lengths of the 5' and 3' homologous arms of the first and second nucleic acid molecules are between about 400 bp and 1,500 bp, between about 500 bp and 1,300 bp, or between about 600 bp and 1,000 bp. In some implementations, the lengths of the 5' and 3' homologous arms of the first and second nucleic acid molecules are between approximately 600 bp and 1,000 bp.

In some implementations, the template sequence length is at least 25 kilobase pairs (KB), at least 50 KB, at least 100 KB, at least 200 KB, at least 400 KB, at least 500 KB, at least 600 KB, at least 700 KB, at least 800 KB, at least 900 KB, at least 1 megabase pair (MB), at least 2 MB, at least 3 MB, at least 4 MB, at least 5 MB, at least 6 MB, at least 7 MB, at least 8 MB, at least 9 MB, at least 10 MB, at least 15 MB, at least 20 MB, at least 25 MB, at least 30 MB, at least 40 MB, at least 50 MB, at least 60 MB, at least 70 MB, at least 80 MB, at least 90 MB, at least 100 MB, at least 120 MB, at least 140 MB, at least 160 MB, at least 180 MB, at least 200 MB, at least 220 MB, or at least 250 MB. In some implementations, the template sequence length is between 50KB and 250MB, 50KB and 100MB, 50KB and 50MB, 50KB and 20MB, 50KB and 10MB, 50KB and 5MB, 50KB and 3MB, 50KB and 2MB, 50KB and 1MB, 100KB and 200MB, 100KB and 100MB, 100KB and 50MB, and 100KB. Between 20MB and 100KB, between 10MB and 10MB, between 100KB and 5MB, between 100KB and 3MB, between 100KB and 2MB, between 100KB and 1MB, between 100KB and 500KB, between 200KB and 10MB, between 200KB and 50MB, between 200KB and 20MB, between 200KB and 10MB, between 200KB and 5MB, between 200KB and 3MB, between 200KB and 2MB Between 200KB and 1MB, between 200KB and 500KB, between 500KB and 100MB, between 500KB and 50MB, between 500KB and 20MB, between 500KB and 10MB, between 500KB and 5MB, between 500KB and 3MB, between 500KB and 2MB, between 500KB and 1MB, between 1MB and 100MB, between 1MB and 50MB, between 1MB and 20MB, between 1MB and 10MB Between 1MB and 5MB, between 1MB and 3MB, between 1MB and 2MB, between 3MB and 100MB, between 3MB and 50MB, between 3MB and 20MB, between 3MB and 10MB, between 3MB and 5MB, between 5MB and 100MB, between 5MB and 50MB, between 5MB and 20MB, between 5MB and 10MB, between 10MB and 100MB, between 10MB and 50MB, or between 10MB and 20MB. In some implementations, the template sequence length is between 200KB and 50MB, between 1MB and 20MB, between 1MB and 10MB, between 1MB and 5MB, between 1MB and 3MB, between 3MB and 20MB, between 3MB and 10MB, between 3MB and 7MB, or between 3MB and 5MB.

In some embodiments, generating a double-strand break at (c) involves inducing the double-strand break using a CRISPR/Cas endonuclease and one or more guide nucleic acids (gNA), one or more zinc finger nucleases, one or more transcription activator-like effector nucleases (TALEN), or one or more CRE recombinases. In some embodiments, the CRISPR/Cas endonuclease includes CasI, CasIB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, CasX, CasY, Cpf1 (Cas12a), Cas12b, Cas13a, CsyI, Csy2, Csy3, CseI, Cse2, CscI, Csc2, Csa5, Csn2, Csm2, Cs CRISPR/Cas endonucleases include m3, Csm4, Csm5, Csm6, CmrI, Cmr3, Cmr4, Cmr5, Cmr6, CsbI, Csb2, Csb3, Csx17, CsxI4, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, CsfI, Csf2, Csf3, Csf4, Cms1, C2c1, C2c2, or C2c3, or their homologs, orthologs, or modified forms. In some embodiments, CRISPR/Cas endonucleases include Cas9, Cpf1 (Cas12a), Cas12b, CasX, CasY, C2c1, or C2c3, or their homologs, orthologs, or modified forms. In some embodiments, CRISPR/Cas endonucleases include Cas9. In some implementations, gNA includes a single guide RNA (sgRNA).

In some embodiments, the target chromosome comprises, from 5' to 3', the 5' homologous arm sequence of the first nucleic acid molecule, the target sequence, and the 3' homologous arm sequence of the second nucleic acid molecule. In some embodiments, the template chromosome comprises, from 5' to 3', the 3' homologous arm sequence of the first nucleic acid molecule, the template sequence, and the 5' homologous arm sequence of the second nucleic acid molecule.

In some embodiments, the target sequence comprises at least 1 gene, at least 2 genes, at least 3 genes, at least 5 genes, at least 10 genes, at least 20 genes, at least 30 genes, at least 40 genes, at least 50 genes, at least 100 genes, or at least 200 genes. In some embodiments, the target sequence comprises one or more genes homologous to one or more genes in the template sequence.

In some embodiments, the template sequence comprises a naturally occurring sequence. In some embodiments, the template sequence comprises at least one gene, at least two genes, at least three genes, at least five genes, at least ten genes, at least twenty genes, at least thirty genes, at least forty genes, at least fifty genes, at least one hundred genes, or at least two hundred genes. In some embodiments, the template sequence comprises one or more modifications to the naturally occurring sequence. In some embodiments, the template sequence comprises an artificial sequence. In some embodiments, the artificial sequence comprises a sequence encoding one or more antibodies or antigen-binding fragments thereof. In some embodiments, one or more antibodies or antigen-binding fragments thereof comprise scFv, bispecific antibodies, or multispecific antibodies.

In some embodiments, the target sequence is deleted by inserting a template sequence. In some embodiments, (a) the target chromosome from 5' to 3' comprises the 5' homologous arm sequence of a first nucleic acid molecule, a first sgRNA target sequence, a target sequence, a second sgRNA target sequence, and the 3' homologous arm sequence of a second nucleic acid molecule; and (b) the template chromosome from 5' to 3' comprises the third sgRNA target sequence, the 3' homologous arm sequence of the first nucleic acid molecule, the template sequence, the 5' homologous arm sequence of the second nucleic acid molecule, and the fourth sgRNA target sequence. In some embodiments, generating a double-strand break includes contacting the cell with a CRISPR/Cas endonuclease and the first, second, third, and fourth sgRNAs. In some embodiments, the first, second, third, and fourth sgRNAs contain targeting sequences specific to the first, second, third, and fourth sgRNA target sequences.

In some implementations, contacting cells with CRISPR/Cas endonucleases and sgRNA involves transfecting cells with one or more nucleic acid molecules encoding CRISPR/Cas endonucleases and sgRNA.

In some embodiments, inserting a template sequence includes deleting a sequence with minimal or no deletion of the target sequence. In some embodiments, inserting a template sequence disrupts one or more functions of the target sequence. In some embodiments, inserting a template sequence disrupts a gene in the target sequence. In some embodiments, (a) the target chromosome from 5' to 3' contains a 5' homologous arm sequence of a first nucleic acid molecule, a first sgRNA target sequence, and a 3' homologous arm sequence of a second nucleic acid molecule; and (b) the template chromosome from 5' to 3' contains a second sgRNA target sequence, a 3' homologous arm sequence of the first nucleic acid molecule, a template sequence, a 5' homologous arm sequence of the second nucleic acid molecule, and a third sgRNA target sequence. In some embodiments, generating a double-strand break includes contacting the cell with a CRISPR/Cas endonuclease and the first, second, and third sgRNAs. In some embodiments, the first, second, and third sgRNAs contain targeting sequences specific to the first, second, and third sgRNA target sequences. In some implementations, contacting cells with CRISPR/Cas endonucleases and sgRNA involves transfecting cells with one or more nucleic acid molecules encoding CRISPR/Cas endonucleases and sgRNA.

In some embodiments, the first or second marker comprises a fluorescent protein operatively linked to a promoter capable of expressing a fluorescent protein in the cell. In some embodiments, the fluorescent protein includes green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), dsRed, mCherry, or tdTomato. In some embodiments, the fluorescent protein includes GFP. In some embodiments, the first marker further includes a selection marker. In some embodiments, the second marker further includes a selection marker. In some embodiments, the selection marker is selected from the group consisting of: dihydrofolate reductase (DHFR), glutamine synthase (GS), puromycin acetyltransferase, blastcin deaminase, histamine dehydrogenase, hygromycin phosphotransferase (hph), bleomycin resistance gene, and aminoglycoside phosphotransferase (neomycin resistance). In some embodiments, the first and second markers are not the same selection marker. In some embodiments, the first marker comprises GFP operatively linked to a promoter capable of expressing GFP in the cell and puromycin acetyltransferase, and the second marker comprises hygromycin phosphotransferase.

In some embodiments, the method further includes (e) deleting all or part of the first or second marker after step (d). In some embodiments, deletion of the first or second marker includes deletion induced by a CRISPR/Cas endonuclease and gNA, wherein the gNA contains a sequence-specific targeting sequence encoding the marker.

In some embodiments, the cells include hybrid cells, embryonic hybrid stem cells (EHS), or fertilized eggs. In some embodiments, EHS cells are generated by fusing ES cells from any two species selected from the group consisting of: mice, rats, rabbits, guinea pigs, hamsters, sheep, goats, donkeys, cattle, horses, camels, chickens, and monkeys. In some embodiments, EHS cells are generated by fusing human embryonic stem cells with embryonic stem cells from a non-human species. In some embodiments, the non-human species are mice, rats, rabbits, guinea pigs, hamsters, sheep, goats, donkeys, cattle, horses, camels, chickens, or monkeys. In some embodiments, EHS cells are generated by fusing EH cells from any two different species selected from the group consisting of: mice, rats, rabbits, guinea pigs, hamsters, sheep, goats, donkeys, cattle, horses, camels, chickens, and monkeys. In some embodiments, the fusion includes electrofusion, viral-induced fusion, or chemically induced fusion.

In some embodiments, the cells include hybrid cells. In some embodiments, generating hybrid cells includes: (a) generating micronucleated human cells; and (b) fusing micronucleated human cells with cells from a non-human species to generate hybrid cells. In some embodiments, micronucleated human cells are generated by exposing human cells to colchicine under conditions sufficient to induce micronucleation and collecting the micronucleated cells using centrifugation. In some embodiments, the non-human species is a mouse, rat, rabbit, guinea pig, hamster, sheep, goat, donkey, cow, horse, camel, chicken, or monkey. In some embodiments, the cells from the non-human species are ES cells, and the hybrid cells are EHS cells.

In some embodiments, the target sequence comprises a gene encoding an immunoglobulin or a T-cell receptor subunit. In some embodiments, the target chromosome includes mouse chromosome 12, and the template chromosome includes human chromosome 14. In some embodiments, the target sequence comprises a mouse Igh variable region sequence. In some embodiments, the mouse Igh variable region sequence comprises sequences encoding segments of the mouse VH, DH, and JH1-6 gene regions and intercalated non-coding sequences. In some embodiments, the template sequence comprises a human IGH variable region sequence. In some embodiments, the human IGH variable region sequence comprises sequences encoding segments of the human VH, DH, and JH1-6 gene regions and intercalated non-coding sequences. In some embodiments, the target sequence comprises a mouse Igl variable region sequence. In some embodiments, the target sequence comprises a mouse Igk variable region sequence. In some embodiments, the template sequence comprises a human IGL variable region sequence. In some embodiments, the template sequence comprises a human IGK variable region sequence. In some embodiments, the mouse Igk variable region sequence comprises sequences encoding segments of the mouse _Vk and _Jk1-5 gene regions and intercalated non-coding sequences. In some embodiments, the template sequence comprises a human IGK variable region sequence. In some embodiments, the human IGK variable region sequence comprises sequences encoding human _Vk and _Jk1-5 gene segments and interpolated non-coding sequences.

In some embodiments, the method further includes recovering engineered chromosomes from the cells selected in step (d). In some embodiments, recovering engineered chromosomes includes exposing cells to colchicine under conditions sufficient to induce micronucleation, and collecting micronucleated cells using centrifugation.

In some implementations, the first and second nucleic acid molecules are plasmids.

This disclosure provides engineered chromosomes produced by the methods of this disclosure.

In some embodiments, the engineered chromosome is mouse chromosome 12 containing a sequence of the human IGH variable region replacing the mouse Igh variable region. In some embodiments, the mouse Igh variable region contains VH, DH, and JH1-6 gene segments and intercalated non-coding sequences. In some embodiments, the human IGH variable region contains VH, DH, and JH1-6 gene segments and intercalated non-coding sequences. In some embodiments, the engineered chromosome is mouse chromosome 6 containing a sequence of the human IGK variable region replacing the mouse Igk variable region. In some embodiments, the mouse Igk variable region sequence contains sequences encoding mouse _Vk and _Jk1-5 gene segments and intercalated non-coding sequences. In some embodiments, the template sequence contains a human IGK variable region sequence. In some embodiments, the human IGK variable region sequence contains sequences encoding human _Vk and _Jk1-5 gene segments and intercalated non-coding sequences.

This disclosure provides cells comprising engineered chromosomes of this disclosure.

In some embodiments, the cells are capable of hybridizing with mouse ES cells. In some embodiments, the cells are embryonic stem (ES) cells, embryonic hybrid stem (EHS) cells, or zygote cells. In some embodiments, the EHS cells are hybrids of human and mouse ES cells. In some embodiments, the ES cells are mouse ES cells. In some embodiments, the cells are micronucleated cells.

This disclosure provides a method for generating mouse embryonic stem cells, comprising: (a) fusing micronucleated cells containing engineered chromosomes generated by any of the methods of this disclosure with mouse ES cells, wherein: (i) the mouse ES cells contain chromosomes homologous to the engineered chromosomes, the homologous chromosomes containing a first fluorescent protein operatively linked to a promoter capable of expressing a fluorescent protein in the ES cells; and (ii) at least one subpopulation of micronucleated cells containing engineered chromosomes, wherein the engineered chromosomes contain a second fluorescent protein different from the first fluorescent protein, the second fluorescent protein being operatively linked to a promoter capable of expressing a fluorescent protein in the ES cells; (b) selecting ES cells expressing the first and second fluorescent proteins; (c) culturing the ES cells selected in step (c) until at least one subpopulation of ES cells loses the homologous chromosome; and (d) selecting ES cells expressing the second fluorescent protein but not the first fluorescent protein.

In some embodiments, culturing cells in step (c) includes culturing cells for at least 5 days, at least 7 days, at least 10 days, or at least 14 days. In some embodiments, selecting cells in steps (b) and (d) includes fluorescence-activated cell sorting (FACS).

This disclosure provides mouse ES cells generated by the methods of this disclosure.

This disclosure provides a transgenic mouse produced from the mouse ES cells of this disclosure.

In some embodiments, generating transgenic mice includes injecting ES cells into diploid blastocysts, nuclear transfer from ES cells to enucleated mouse embryos, or complementation of tetraploid embryos. In some embodiments, mouse chromosome 12 contains a sequence representing the human IGH variable region replacing the mouse Igh variable region. In some embodiments, the mouse Igh variable region contains VH, DH, and JH1-6 gene segments and intercalated non-coding sequences. In some embodiments, the human IGH variable region contains VH, DH, and JH1-6 gene segments and intercalated non-coding sequences. In some embodiments, mouse chromosome 6 contains a sequence representing the human IGK variable region replacing the mouse Igk variable region. In some embodiments, the mouse Igk variable region sequence contains sequences encoding mouse _Vk and _Jk1-5 gene segments and intercalated non-coding sequences. In some embodiments, the template sequence contains a human IGK variable region sequence. In some embodiments, the human IGK variable region sequence contains sequences encoding human _Vk and _Jk1-5 gene segments and intercalated non-coding sequences.

This disclosure provides a method for generating antibodies, comprising: (a) attacking a transgenic mouse of the present disclosure with an antigen, thereby generating a variety of antibodies comprising human V, D and J segments from the human IGH variable region; and (b) isolating antibodies specific to the antigen.

This disclosure provides a method for generating antibodies, comprising: (a) attacking a transgenic mouse of the present invention with an antigen, thereby generating a variety of antibodies, said antibodies comprising human V and J segments from the human IGK or IGL variable region; and (b) isolating antibodies specific to the antigen.

This disclosure provides antibodies derived from the transgenic mice of this disclosure. In some embodiments, the antibody comprises a single-chain variable segment (scFv), a bispecific antibody, or a multispecific antibody.

This disclosure provides a method for generating chromosomal rearrangements, comprising: (a) providing cells containing a target chromosome with a target location and a template chromosome containing a template sequence; (b) contacting the cells with a nucleic acid molecule comprising a 5' homologous arm and a 3' homologous arm from 5' to 3', the 5' homologous arm containing a nucleotide sequence upstream of the 5' end of the target location, and the 3' homologous arm containing a nucleotide sequence upstream of the 5' end of the template sequence; (c) generating a double-strand break at the target location and at the 5' end of the template sequence, thereby inserting a marker into the target chromosome at the 3' of the 5' homologous arm sequence, followed by insertion into the template sequence, thereby generating chromosomal rearrangements; and (d) selecting one or more cells expressing the marker.

In some embodiments, the lengths of the 5' and 3' homologous arms of the nucleic acid molecule are between about 20 bp and 2,000 bp, between about 50 bp and 1,500 bp, between about 100 bp and 1,400 bp, between about 150 bp and 1,300 bp, between about 200 bp and 1,200 bp, between about 300 bp and 1,100 bp, between about 400 bp and 1,000 bp, or between about 500 bp and 900 bp, or between about 600 bp and 800 bp. In some embodiments, the lengths of the 5' and 3' homologous arms of the nucleic acid molecule are between about 400 bp and 1,500 bp, between about 500 bp and 1,300 bp, or between about 600 bp and 1,000 bp. In some embodiments, the lengths of the 5' and 3' homologous arms of the nucleic acid molecule are between about 600 bp and 1,000 bp.

In some embodiments, generating double-strand breaks in (c) includes inducing double-strand breaks using a CRISPR/Cas endonuclease and at least one sgRNA, one or more zinc finger nucleases, one or more transcription activator-like effector nucleases (TALENs), or one or more CRE recombinases. In some embodiments, the CRISPR/Cas endonuclease includes CasI, CasIB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, CasX, CasY, Cas12a(Cpf1), Cas12b, Cas13a, CsyI, Csy2, Csy3, CseI, Cse2, CscI, Csc2, Csa5, Csn2, Cs CRISPR/Cas endonucleases include m2, Csm3, Csm4, Csm5, Csm6, CmrI, Cmr3, Cmr4, Cmr5, Cmr6, CsbI, Csb2, Csb3, Csx17, CsxI4, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, CsfI, Csf2, Csf3, Csf4, Cms1, C2c1, C2c2, or C2c3, or their homologs, orthologs, or modified forms. In some embodiments, the CRISPR/Cas endonuclease includes Cas9, Cpf1, CasX, CasY, C2c1, or C2c3, or their homologs, orthologs, or modified forms. In some embodiments, the CRISPR/Cas endonuclease includes Cas9. In some embodiments, generating a double-strand break includes contacting cells with a CRISPR/Cas endonuclease, at least a first gNA and a second gNA, the first gNA containing a target-specific targeting sequence such that the CRISPR/Cas endonuclease cleaves the target site, and the second gNA containing a target sequence specific to the 5' end of the template sequence. In some embodiments, contacting cells with the CRISPR/Cas endonuclease and sgRNA includes transfecting cells with one or more nucleic acid molecules encoding the CRISPR/Cas endonuclease and sgRNA. In some embodiments, the one or more nucleic acid molecules are plasmids.

In some embodiments, the marker comprises a fluorescent protein operatively linked to a promoter capable of expressing a fluorescent protein in the cell. In some embodiments, the fluorescent protein includes GFP, YFP, RFP, CFP, BFP, dsRed, mCherry, or tdTomato. In some embodiments, the marker further includes a selection marker. In some embodiments, the selection marker is selected from the group consisting of: dihydrofolate reductase (DHFR), glutamine synthase (GS), puromycin acetyltransferase, blastcin deaminase, histamine dehydrogenase, hygromycin phosphotransferase (hph), bleomycin resistance gene, and aminoglycoside phosphotransferase (neomycin resistance).

In some implementations, the cells include embryonic stem (ES) cells.

In some implementations, the nucleic acid molecule is a plasmid.

This disclosure provides cells comprising chromosomal rearrangements produced by the methods of this disclosure. In some embodiments, the cells are mouse ES cells.

This disclosure provides transgenic mice derived from mouse ES cells produced by the methods of this disclosure.

Attached Figure Description

A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description and accompanying drawings illustrating the illustrative embodiments, wherein:

Figure 1 is a diagram from top to bottom showing the mouse immunoglobulin heavy chain complex (Igh), human Igh, and the humanized mouse Igh containing variable domains ( _VH , _DH , and _JH1-6 ). Chro: chromosome.

Figure 2 is a diagram illustrating the hybridization of engineered mouse and human embryonic stem (ES) cells via electrofusion. Mouse ES cells express the marker neomycin, while human ES cells express mCherry. Embryonic hybrid stem cells (hybridoma cells) are resistant to G418 and positive for mCherry.

Figure 3A is a diagram showing the placement of three pairs of PCR primers (as indicated by arrows) in the _VH , _DH and _JH1-6 regions of the human Igh gene, which are used for genotyping of embryonic hybrid stem cells (EHS).

Figure 3B is an exemplary gel showing the PCR results of 12 embryonic hybrid stem cell (EHS) clones, which were genotyped using the primers shown in Figure 3A.

Figures 4A-4B are diagrams illustrating the process of establishing engineered humanized chromosomes in EHS cells (Figure 4A) via HDR-mediated chromosome rearrangement (HCMR). HDR: Homologous Directed Repair. EHS cells were co-transfected with the following plasmids: a 5’ HMCR plasmid containing a 5’ arm homologous to the mouse Igh gene, a 3’ arm homologous to the human Igh gene, and a pCMV-EGFP-polyA-PGK-purinemycin-polyA box; a 3’ HMCR plasmid containing a 5’ arm homologous to the 3’ end of the human Igh variable locus, a 3’ arm homologous to the 3’ end of the mouse Igh variable locus, and a PGK-hygromycin-polyA box; and four plasmids containing Cas9 and sgRNAs targeting the 5’ and 3’ variable domains of mouse Igh and human Igh, as shown. Alternatively (Figure 4B) via CRE-Loxp-mediated chromosomal rearrangement (CMCR): Four plasmids were designed to mediate the CMCR process. The mouse Igh 5’ (pCMV-GFP-BGH PolyA-Loxp) and 3’ (BGH polyA-Loxp-511-hygromycin-BGH polyA-PGK-BSD-BGHPolyA) plasmids were designed to insert into the 5’ and 3’ ends of the mouse Igh variable locus, respectively. Simultaneously, the human IGH 5’ (BGHpolyA-Loxp-Puro-BGH PolyA-PGK-neomycin-BGH polyA) and 3’ (pCMV-BGP-BGH polyA-PGK-Loxp-511) plasmids were designed to insert into the 5’ and 3’ ends of the human IGH variable locus, respectively. Crewas was transfected into successfully integrated EHS cells for CMCR.

Figure 5A is a diagram showing the placement of PCR primers (as indicated by arrows) used to verify engineered human chromosomes.

Figure 5B shows the PCR results using the four primer pairs shown in Figure 5A. Results for 192 single clones are displayed.

Figure 6 is a diagram illustrating the replacement of mouse chromosomes with engineered human chromosomes in mouse ES cells. EHS cells carrying engineered human chromosomes labeled with GFP were micronized by exposure to colchicine. Microcells were collected by centrifugation and electrofused with mouse ES cells in which the corresponding mouse chromosomes were labeled with mCherry. GFP+mCherry+ cells were separated by fluorescence-activated cell sorting (FACS). The cells were then cultured, and GFP+mCherry- cells that had lost their mouse chromosomes were separated by FACS.

Figure 7A shows the placement of PCR primers (as indicated by arrows) used to validate Igh humanized mice.

Figure 7B shows the PCR results of an exemplary Igh humanized mouse using the 7 primer pairs shown in Figure 7A.

Figure 8A shows the fluorescence in situ hybridization (FISH) results of Igh humanized mice.

Figure 8B shows the G-banding karyotype analysis of Igh humanized mice.

Figure 9A shows the whole-genome sequencing (WGS) analysis of IGH-V from Igh humanized mice. The copy number of the WGS sequence for each variable (V) gene segment located in the _VH region of human Igh is shown.

Figure 9B shows the WGS analysis of IGH-D and IGH-J in humanized Igh mice. The copy number of the WGS sequence is shown for each diversity (D) gene segment and the six linking (J) segments located in the _DH and _JH1-6 regions of human Igh.

Figure 10 shows the humanization of the variable domain of the mouse Igk gene.

Figures 11A-11B show the PCR validation results for Igk humanized mice. Figure 11A shows the positions of the primers designed for the PCR experiments. Figure 11B shows the PCR results for Igk humanized mice using the 5 primer pairs listed in Figure A.

Figure 12 shows the WGS analysis results of Igk humanized mice. Copy number of each antibody gene located in the _VK and _Jk regions of the human IGK gene in the WGS sequence.

Detailed Implementation

This disclosure provides methods for engineering chromosomes, which include transferring large sequence fragments between chromosomes. Using the methods disclosed herein, at least 5 trillion pairs (MB) of sequences can be transferred from a non-achromosomal template to a target chromosome. The methods disclosed herein can also be used to generate chromosomal rearrangements, such as inversions and translocations. This document also provides engineered chromosomes generated by the methods of this disclosure, as well as cells and animals containing these engineered chromosomes, and methods for using them.

Manipulating large segments of genes or chromosomes holds immense promise for basic and translational research and the development of therapies. Genetic humanization is one of the most popular applications, where genes in model organisms such as mice are replaced with their human counterparts. For example, mice carrying humanized Ig genes provide a powerful platform for generating human antibodies in a mouse context. However, manipulating large segments remains one of the most significant challenges in gene editing because delivery vectors capable of carrying up to one million base pairs (MB) of chromosome fragments are unavailable. The payload capacity of conventional delivery vectors, such as adeno-associated virus vectors or other viral vectors, is limited by the size of the viral genome from which the vector originates.

The methods disclosed in this paper allow for efficient in-situ substitution of large sequences between chromosomes. These methods, known as Massive Fragment Across Species In Situ Replacement Technology (MASIRT), can be used to replace large portions of a chromosome in a single editing step, and in some cases, sequences up to megabase pairs (MB). These methods can be used to efficiently transfer large sequences between species or between chromosomes of a single species. In one example, MASIRT was used to obtain humanized mice with variable domains targeting the mouse Igh gene. Humans and mice exhibit high similarity in the arrangement and expression of antibody genes, and the genomic structure of heavy chains is also similar across these species. Therefore, using MASIRT, approximately 3 MB of mouse genome sequence containing all _VH , _DH , and _JH gene segments was replaced with approximately 1 Mb of continuous human genome sequence containing equivalent human gene segments, resulting in the humanized mouse Igh gene.

Unlike other methods that target only embryonic stem cells, the method disclosed herein can be advantageously used to replace large sequences in fertilized eggs. Embryonic stem cell lines are generally not applicable to species other than mice. Instead, fertilized eggs are available in many mammals, thus the method disclosed herein can be used to obtain animals with humanized genes or gene fragments, such as rabbits or cattle. Furthermore, the method disclosed herein can be used to replace large sequence fragments, such as sequences up to at least 5 MB, in a single replacement, approximately five times faster than other methods known in the art. This increases efficiency and reduces the time and cost required to produce animals with humanized genes. For example, Igh humanized mice can be produced with only three rounds of replacement. Another advantage is that, when used in mice, each replacement takes only 1-3 months, which is only half or one-third the time required by other methods known in the art.

definition

Chromosomes are long DNA molecules that contain all or part of an organism's genetic material. Most eukaryotic chromosomes include packaging proteins called histones, which, with the help of chaperone proteins, bind to and compress the DNA molecule to maintain its integrity. Eukaryotic chromosomes consist of long, linear DNA molecules associated with proteins, forming a tight complex of proteins and DNA called chromatin. Each chromosome has a centromere, from which one or two arms extend. The arms of a chromosome terminate at telomeres, regions of repetitive nucleotide sequences associated with specialized proteins that protect the ends of chromosomal DNA from progressive degradation and ensure the integrity of linear chromosomes by preventing the DNA repair system from mistaking the very ends of the DNA strand for double-strand breaks.

A "gene" includes a DNA region that encodes a gene product (e.g., a protein or non-coding RNA) and all DNA regions that regulate the production of the gene product, regardless of whether such regulatory sequences are adjacent to coding and/or transcribed sequences. Therefore, a gene may include regulatory element sequences, including, but not limited to, promoter sequences, terminators, translation regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions. The coding sequence encodes the gene product during transcription or transcription and translation. The coding sequences of this disclosure may comprise fragments and do not necessarily contain a full-length open reading frame. A gene may include the transcribed strand and a complementary strand containing anticodons. A gene may also include exons (which may include protein-coding sequences and untranslated regions) and introns (which are removed from the final RNA product by splicing).

As used herein, the term "promoter" can refer to a DNA sequence located adjacent to the DNA sequence encoding a recombinant product. A promoter is preferably effectively linked to an adjacent DNA sequence. A promoter typically increases the amount of protein or RNA product expressed from a DNA sequence compared to the amount expressed in the absence of a promoter. Promoters from one organism can be used to enhance protein expression from DNA sequences derived from another organism. For example, vertebrate promoters can be used to express GFP from jellyfish in vertebrates. Furthermore, a promoter element can increase the amount of recombinant product expressed from multiple DNA sequences linked in tandem. Thus, a promoter element can enhance the expression of one or more recombinant products. Multiple promoter elements are well known to those skilled in the art.

As used herein, the term "enhancer" can refer to a DNA sequence located adjacent to or distal to the DNA sequence encoding a protein or RNA product. Enhancer elements are typically located upstream of promoter elements, but can also be located downstream of or within the coding DNA sequence, such as within introns. In some cases, enhancers can be located thousands, tens, or even hundreds of kilobases away from the gene they regulate. Enhancer elements can increase the amount of protein or RNA product expressed from the DNA sequence beyond the increase provided by promoter elements. Various enhancer elements are readily available to those skilled in the art.

As used herein, the terms “exogenous chromosome” or “exogenous sequence” refer to a foreign chromosome or sequence relative to the animal genome. For example, in mouse cells (where all chromosomes except one human chromosome are mouse chromosomes), human chromosomes are exogenous chromosomes. Similarly, in mouse chromosomes where a portion of the mouse sequence has been replaced by a human sequence, the human sequence is referred to as an exogenous sequence. Similarly, “endogenous” refers to chromosomes or sequences derived from an organism, such as the mouse chromosomes or sequences described above.

As used herein, the term “homologous recombination” refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules, called homologous sequences or homologous arms. Homologous recombination typically involves the following basic steps: After a double-strand break (DSB) occurs on both DNA strands, a segment of DNA around the 5’ end of the DSB is removed in a process called excision. In the subsequent strand invasion step, the overhanging 3’ end of the broken DNA molecule “invades” an unbroken similar or identical (or homologous) DNA molecule, such as a homologous arm. After strand invasion, the further sequence of events can follow either of two pathways—the DSBR (double-strand break repair) pathway or the SDSA (synthesis-dependent strand annealing) pathway.

As used herein, “DNA repair pathways” refer to cellular mechanisms that allow cells to maintain genomic integrity in response to DNA damage, such as the detection of single-strand or double-strand breaks in DNA. Depending on the type and extent of DNA damage, and the stage of the cell cycle, DNA repair pathways may include, but are not limited to, excision, canonical homology-directed repair (canonical HDR), homology recombination (HR), alternative homology-directed repair (alt-HDR), double-strand break repair (DSBR), single-strand annealing (SSA), synthesis-dependent strand annealing (SDSA), break-induced replication (BIR), alternative end joining (alt-EJ), microhomology-mediated end joining (MMEJ), and DNA synthesis-dependent microhomology-mediated end joining (S… D-MMEJ), non-homologous end joining (NHEJ) pathways, such as canonical non-homologous end joining (C-NHEJ) repair, alternative non-homologous end joining (A-NHEJ) pathway, trans-damage DNA synthesis (TLS) repair, base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), DNA damage response (DDR), blunt end joining, single strand break repair (SSBR), interstrand crosslink repair (ICL), and the Fanconi anemia pathway (FA).

As used in this article, homology-directed repair (HDR) refers to the process of repairing DNA damage using homologous nucleic acids (e.g., sister chromatids or exogenous nucleic acids). In normal cells, HDR typically involves a series of steps such as identifying breaks, stabilizing breaks, excision, stabilizing single-stranded DNA, forming DNA cross-linking intermediates, splitting cross-linking intermediates, and joining.

As used herein, a “homologous” is a protein within a group of proteins that perform the same biological function, such as proteins belonging to the same protein family and providing common traits or performing the same or similar biological functions. Homologous proteins are expressed by homologous genes. A homologous gene is a gene that encodes a protein that has the same or similar biological function as a protein encoded by a second gene. Homologous genes can arise through speciation events (orthologous proteins) or through genetic replication events (paralogous proteins). An “orthologous protein” is a group of homologous genes that evolved from a common ancestral gene in different species through speciation. Normally, orthologous proteins maintain the same function during evolution. A “paralogous protein” is a group of homologous genes in the same species that diverge from each other due to gene replication. Therefore, homologous genes can originate from the same or different organisms. Homologous genes include naturally occurring alleles and artificially generated variants. The percentage of identity between homologous proteins will depend on the origin of the protein and the degree of divergence between the species from which the protein originated. Homologous proteins from more closely related species (e.g., two mammals such as humans and mice) are generally more similar than proteins from more distantly related species (e.g., chickens and mice). When optimally aligned, homologous proteins typically share at least about 40%, about 50%, or about 60% similarity across the entire protein length, and in some cases at least about 70%, such as about 80%, or even at least about 90%. In other cases, such as when comparing proteins from highly divergent species, homologous proteins will share at least about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% similarity along the length of conserved protein domains (such as DNA-binding domains).

Homologous genes or proteins are identified by comparing DNA or amino acid sequences, either manually or using computer-based tools. These computer-based tools employ known homology-based search algorithms, such as those commonly known as BLAST, FASTA, and Smith-Waterman. Local sequence alignment procedures (e.g., BLAST) can be used to search sequence databases for similar sequences, and a summary expectation value (E-value) is used to measure sequence base similarity. Because a protein hit with the best E-value for a particular organism may not necessarily be an ortholog (i.e., having the same function) or a unique ortholog, a reciprocal query can be used to filter hit sequences with significant E-values for ortholog identification. A reciprocal query requires searching a database of amino acid sequences from the basal organism for significant hits similar to the query protein sequence. A hit can be identified as an ortholog when the best hit of the reciprocal query is the query protein itself or a protein encoded by a gene that replicates after speciation.

As used herein, “identity percentage” refers to the degree to which two optimally aligned DNA or protein segments remain unchanged throughout an alignment window containing the entire component (e.g., nucleotide or amino acid sequence). The “identity score” of the aligned segments of the test and reference sequences is the number of common components shared by the sequences of the two aligned segments divided by the total number of sequence components in the reference segment within the alignment window, which is the smaller of the complete test or complete reference sequences. The “identity percentage” (“identity %”) is the identity score multiplied by 100. This optimal alignment is understood to be considered a local alignment of the DNA sequence. For protein alignment, local alignment of the protein sequence should allow for the introduction of gaps to achieve optimal alignment. The identity percentage can be calculated over the alignment length excluding gaps introduced by the alignment itself.

As used herein, “specific to” when referring to a nucleotide sequence such as the homologous arm of a guide RNA or a target sequence means a sequence that is identical or substantially identical to another nucleotide sequence or its inverse complementary sequence. A sequence “specific to” another sequence is capable of hybridizing with the other sequence or its inverse complementary sequence via Worson-Crick base pairing. Therefore, those skilled in the art will understand that a sequence specific to another sequence is highly similar to the other sequence or its inverse complementary sequence, but not necessarily identical. For example, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with another sequence remains specific to that sequence if it can hybridize with it. As another example, a guide nucleic acid target sequence may contain one, two, three, or more mismatches with the target sequence, depending on the location of the mismatch in the target sequence, and it remains specific to the target sequence if it can target a ribonucleoprotein complex containing gNA and an endonuclease to the target sequence.

As used herein, “selection” refers to separating a population of two distinct products using any method known in the art. When applied to cells, chromosomes, or sequences, selection can be based on markers such as selection markers. Selecting cells expressing a selection marker involves culturing a mixed cell population comprising cells expressing the marker and cells not expressing the marker in a selective medium, thereby killing cells not expressing the marker or inhibiting their growth. They can be similarly selected by placing a marker-containing sequence or chromosome within cells and applying a selection protocol. Similarly, selection can be based on detection markers (such as fluorescent proteins). Methods known in the art, such as fluorescence-activated cell sorting (FACS), can be used to physically remove cells expressing the detection marker from a mixed cell population based on the detection marker. Optionally, or additionally, the mixed cell population can be diluted to allow the isolation and culture of single cells, and the presence of one or more traits, such as markers, of clones derived from the isolated cells can be determined.

As used herein, “derived from” refers to the source or origin of a molecular entity, such as a nucleic acid or protein. The source of a molecular entity can be a naturally occurring, recombinant, unpurified, or purified molecular entity. For example, a polypeptide derived from a second polypeptide may contain an amino acid sequence that is identical or substantially similar to that of a second protein, such as having more than 50% homology. The derived molecular entity, such as a nucleic acid or protein, may contain one or more modifications, such as one or more amino acid or nucleotide changes.

"Separated from" refers to a molecular entity that has been purified, removed, or separated from its source or origin.

A “naturally occurring” sequence is a sequence found in at least one species that exists in nature.

"Artificial sequences" are sequences that do not exist in nature. Artificial sequences may resemble natural sequences, but contain one or more alterations relative to naturally occurring sequences. Optionally, artificial sequences may have little or no similarity to any naturally occurring sequence. Chimeric or recombinant sequences are a class of artificial sequences in which two sequences from different sources, or two sequences that have never been found to be adjacent to each other, are operatively joined together.

"Operatively linked" or "operably linked" refers to the juxtaposition of genetic elements in a relationship that allows them to function in the intended manner. For example, if a promoter facilitates the initiation of transcription of a coding sequence, then the promoter is effectively linked to the coding region. Intercalated residues may exist between the promoter and the coding region as long as this functional relationship is maintained.

This document uses the following classification to refer to stem cells. In terms of developmental stage, the most pluripotent and earliest are “embryonic stem (ES) cells” or “ES cells.” ES cells can be primary cells of fresh origin or derived from ES cell lines. All other stem cells derived from somatic tissues (every tissue except germ cell tissues) are broadly defined as “somatic stem cells,” but may generally be referred to as any or all of the following: “adult stem cells,” “mature stem cells,” “progenitor cells,” “precursor cells,” and “precursor stem cells.” Another class of non-embryonic stem cells is defined as “germline stem cells.” Finally, this document describes non-stem cells as “mature cells,” but also refers to them as “differentiated cells,” “mature differentiated cells,” “terminally differentiated cells,” and “somatic cells.” Mature cells can also be primary isolated cells derived from tissues or immortalized cell lines or tumor-derived cell lines. This invention also includes “precursor forms of mature cells,” which include all cells that do not conform to the commonly used scientific definitions of stem cells or mature cells. ES cells can be cultured in vitro for extended periods and induced to revert to the normal program of embryonic development to differentiate into all cell types of adult animals, including germ cells, before being inserted/injected into the lumen of a normal blastocyst.

As used herein, a "hybrid cell" refers to a cell containing elements from two genomes. Those skilled in the art will understand that a hybrid cell may contain two complete or nearly complete genomes from different sources. Alternatively, a hybrid cell may contain a complete genome from one source and only a few chromosomes, one chromosome, or a portion of one chromosome from a second source. Cells containing any mixture of elements from the two genomes between these two extremes are still considered hybrid cells. The two genomes in a hybrid may originate from different individuals, different strains of the same species, or different species. Hybrid cells can be produced by any method known in the art. These techniques include, but are not limited to, cell fusion and microcell-mediated chromosome transfer (MMCT), which involves transferring a small number of chromosomes from one cell to another.

As used in this article, “hybrid embryonic stem (EHS)” cells refer to hybrid cells that possess embryonic stem cell characteristics. EHS cells can be generated by the fusion of ES cells from two different species, or by MMCT-mediated chromosome transfer from cells of one species to stem cells of another species.

As used herein, “cancer” refers to a disease, ailment, trait, genotype, or phenotype characterized by unregulated cell growth or replication known in the art. Cancer includes solid tumors and liquid tumors. Exemplary cancers include, but are not limited to, leukemia, breast cancer, bone cancer, brain cancer, head and neck cancer, retinal cancer, esophageal cancer, gastric cancer, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, colorectal cancer, lung cancer, bladder cancer, prostate cancer, lung cancer (including both small cell and non-small cell lung cancer), pancreatic cancer, sarcoma, cervical cancer, head and neck cancer, and skin cancer.

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated as incorporated by reference.

Methods for engineered chromosomes

This disclosure provides a method for engineering chromosomes using a template chromosome, a target chromosome, one or more nucleic acid molecules such as vectors or plasmids, and homology-directed repair. A nuclease is used to generate double-strand breaks, located flanking a template sequence in the template chromosome and flanking a target sequence or at a target location in the target chromosome. One or more nucleic acid molecules comprising a marker and homologous arms are used to guide the replacement of the target sequence with the template sequence, the insertion of the template sequence at the target location, or the generation of chromosomal rearrangements by connecting the target and template sequences at double-strand break sites, wherein the homologous arms contain sequences of both the target and template chromosomes.

In some implementations, the method includes replacing the target sequence with a template sequence, i.e., deleting the target sequence by inserting a template sequence.

In some implementations, the method includes replacing the target sequence with a template sequence. Any suitable template sequence and any suitable target sequence can be used in the methods described herein. For example, the method can be used to replace a portion of a chromosome in a model organism with a homologous human sequence, thereby humanizing that portion of the model organism's genome. Alternatively, a large sequence can be inserted at the target site with little or no deletion of the target sequence.

In some embodiments, this disclosure provides a method for generating engineered chromosomes, comprising: (a) providing cells containing a target chromosome containing a target sequence and a template chromosome containing a template sequence; (b) contacting the cells with (i) a first nucleic acid molecule and (ii) a second nucleic acid molecule, the first nucleic acid molecule comprising, from 5' to 3', a 5' homologous arm, at least a first marker, and a 3' homologous arm, the 5' homologous arm containing a nucleotide sequence upstream of the 5' end of the target sequence, and the 3' homologous arm containing a nucleotide sequence upstream of the 5' end of the template sequence; the second nucleic acid molecule comprising, from 5' to 3', a 5' homologous arm, at least a second marker, and a 3' homologous arm, the 5' homologous arm containing a nucleotide sequence downstream of the 3' end of the template sequence, and the 3' homologous arm containing a nucleotide sequence downstream of the 3' end of the target sequence; (c) generating double-strand breaks on either side of the target sequence and at the 5' and 3' ends of the template sequence, thereby inserting the template sequence and the first and second markers into the target chromosome; and (d) selecting one or more cells expressing the first and second markers. In some embodiments, the first and/or second nucleic acid molecule is a plasmid. For some embodiments of the method described herein, the arrangement of the template sequence, target sequence, and homologous arms of the first and second nucleic acid molecules is shown in Figures 4A-4B. In some embodiments, after inserting the template sequence, the first marker is located at the 5' end of the template sequence, and the second marker is located at the 3' end of the template sequence. For example, the engineered chromosome produced by the method described herein, after inserting the template sequence and deleting the target sequence, includes, from 5' to 3', the target chromosome sequence upstream of the target sequence, the first marker, the template sequence, the second marker, and the target chromosome sequence downstream of the target sequence.

Skilled technicians will understand that template sequences of many lengths are suitable for the methods described herein. Suitable template sequences can be as small as a few hundred base pairs or contain a large portion of a chromosome, thus reaching lengths of hundreds of mega-pairs. In some embodiments of the methods described herein, the template sequence length is at least 25 KB, at least 50 KB, at least 100 KB, at least 200 KB, at least 400 KB, at least 500 KB, at least 600 KB, at least 700 KB, at least 800 KB, at least 900 KB, at least 1 MB, at least 2 MB, at least 3 MB, at least 4 MB, at least 5 MB, at least 10 MB, at least 15 MB, at least 20 MB, at least 50 MB, at least 100 MB, at least 150 MB, at least 200 MB, or at least 250 MB. In some implementations, the length of the template sequence is between 50KB and 250MB, between 100KB and 200MB, between 200KB and 50MB, between 500KB and 50MB, between 1MB and 100MB, between 1MB and 10MB, between 1MB and 5MB, between 1MB and 3MB, between 5MB and 50MB, between 5MB and 10MB, between 3MB and 10MB, or between 5MB and 50MB.

In some embodiments of the method described herein, the template chromosome comprises, from 5' to 3', the 3' homologous arm sequence of the first nucleic acid molecule, the template sequence, and the 5' homologous arm sequence of the second nucleic acid molecule. In some embodiments, the template chromosome comprises, from 5' to 3', the 3' homologous arm sequence of the first nucleic acid molecule, the third endonuclease site, the template sequence, the fourth endonuclease site, and the 5' homologous arm sequence of the second nucleic acid molecule.

Skilled technicians will understand that target sequences of many lengths are suitable for the methods described herein. Suitable target sequences can be as small as endonuclease sites (target sites) used to generate double-strand breaks, or encompass a large portion of the chromosome, thus reaching lengths of hundreds of mega-pairs. In some embodiments of the methods described herein, the target sequence length is at least 25 KB, at least 50 KB, at least 100 KB, at least 200 KB, at least 400 KB, at least 500 KB, at least 600 KB, at least 700 KB, at least 800 KB, at least 900 KB, at least 1 MB, at least 2 MB, at least 3 MB, at least 4 MB, at least 5 MB, at least 10 MB, at least 15 MB, at least 20 MB, at least 50 MB, at least 100 MB, at least 150 MB, at least 200 MB, or at least 250 MB. In some implementations, the length of the target sequence is between 50KB and 250MB, 100KB and 200MB, 200KB and 50MB, 500KB and 50MB, 1MB and 100MB, 1MB and 10MB, 1MB and 5MB, 1MB and 3MB, 5MB and 50MB, 5MB and 10MB, 3MB and 10MB, or 5MB and 50MB.

In some embodiments of the method described herein, the target chromosome comprises, from 5' to 3', a 5' homologous arm sequence of a first nucleic acid molecule, a target sequence, and a 3' homologous arm sequence of a second nucleic acid molecule. In some embodiments, the target chromosome comprises, from 5' to 3', a 5' homologous arm sequence of a first nucleic acid molecule, a first endonuclease site, a target sequence, a second endonuclease site, and a 3' homologous arm sequence of a second nucleic acid molecule.

In some embodiments, the nucleic acid molecule used in the methods described herein is a DNA molecule. In some embodiments, the nucleic acid molecule used in the methods described herein is circular, such as a plasmid. Optionally, additional endonuclease sites may be used to linearize the nucleic acid molecules of this disclosure. Exemplary endonuclease sites include, but are not limited to, restriction endonucleases, as well as the CRISPR/Cas endonucleases, ZFN, and TALEN described herein. Those skilled in the art can incorporate suitable endonuclease sites into nucleic acid molecules, such as adjacent to or near any one or two homologous arms of the nucleic acid molecule. Those skilled in the art can integrate suitable CRE recombinase sites into nucleic acid molecules.

In some embodiments, the target sequence is deleted by inserting a template sequence, and the template and target chromosomes are cleaved on either side of the template and target sequences by a CRISPR/Cas ribonucleoprotein. In some embodiments, (a) the target chromosome from 5' to 3' comprises the 5' homologous arm sequence of a first nucleic acid molecule, a first sgRNA target sequence, a target sequence, a second sgRNA target sequence, and the 3' homologous arm sequence of a second nucleic acid molecule; and (b) the template chromosome from 5' to 3' comprises a third sgRNA target sequence, the 3' homologous arm sequence of the first nucleic acid molecule, the template sequence, the 5' homologous arm sequence of the second nucleic acid molecule, and a fourth sgRNA target sequence. In some embodiments, the first, second, third, and fourth sgRNAs contain different target sequences. For example, the first sgRNA contains a targeting sequence specific to the first sgRNA target sequence on the target chromosome, the second sgRNA contains a targeting sequence specific to the second sgRNA target sequence on the target chromosome, the third sgRNA contains a targeting sequence specific to the third sgRNA target sequence on the template chromosome, and the fourth sgRNA contains a targeting sequence specific to the fourth sgRNA target sequence on the target chromosome. Optionally, one or more sgRNA target sequences and the corresponding sgRNA target sequences may be the same sequence.

In some implementations, the inserted template sequence includes a sequence with minimal deletion of the target sequence or a sequence without deletion of the target sequence. Those skilled in the art will understand that many mechanisms of double-strand break repair involve the excision of the break ends, thus creating deletions around the endonuclease sites described herein. For example, deletions of approximately 5 bp, 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, or 50 bp can be generated around the target site or flanking the target sequence via the methods described herein.

In some embodiments (e.g., those in which the target sequence is virtually or not deleted by the methods described herein), (a) the target chromosome from 5' to 3' comprises the 5' homologous arm sequence of the first nucleic acid molecule, the first sgRNA target sequence, and the 3' homologous arm sequence of the second nucleic acid molecule; and (b) the template chromosome from 5' to 3' comprises the second sgRNA target sequence, the 3' homologous arm sequence of the first nucleic acid molecule, the template sequence, the 5' homologous arm sequence of the second nucleic acid molecule, and the third sgRNA target sequence. In some embodiments, the first, second, and third sgRNAs comprise different target sequences. For example, the first sgRNA comprises a target sequence specific to the first sgRNA target sequence on the target chromosome, the second sgRNA comprises a target sequence specific to the second sgRNA target sequence on the target chromosome, and the third sgRNA comprises a target sequence specific to the third sgRNA target sequence on the template chromosome.

In some implementations, inserting a template sequence disrupts one or more functions of the target sequence. For example, inserting a template sequence into the coding sequence of a gene can prevent the expression of the correct gene product by generating premature stop codons, mutations in protein-coding sequences, aberrant splicing products, etc. Similarly, inserting a template sequence into the regulatory sequence of a gene, such as an enhancer or promoter, can prevent gene expression.

In some embodiments, the method of this disclosure includes deleting a first and/or second marker after inserting the target sequence. The marker can be deleted by any suitable method known in the art; for example, a cell containing an engineered chromosome can be contacted with a CRISPR/Cas ribonucleoprotein containing a gNA targeting sequence specific to the sequence encoding the marker, thereby inducing the complete or partial deletion of the marker sequence.

The methods disclosed herein can be used to generate chromosomal rearrangements, such as inversions and translocations. Many chromosomal rearrangements play a role in human diseases or conditions such as cancer. Reconstructing such rearrangements in model organisms (such as mice) can facilitate research on these diseases or conditions. The chromosomal aberrations involved are known to those skilled in the art and are described in the Mitelman database, available at mitelmandatabase.isb-cgc.org/. More information on chromosomal aberrations associated with human diseases is also available at rareiseases.info.nih.gov/diseases/diseases-by-category/36/chromosome-disorders.

Therefore, this disclosure provides a method for generating chromosomal rearrangements, comprising: (a) providing a cell containing a target chromosome with a target location and a template chromosome containing a template sequence; (b) contacting the cell with a nucleic acid molecule containing a 5' homologous arm and a 3' homologous arm from 5' to 3', the 5' homologous arm containing a nucleotide sequence upstream of the 5' end of the target location, and the 3' homologous arm containing a nucleotide sequence upstream of the 5' end of the template sequence; (c) generating a double-strand break at the target location and at the 5' end of the template sequence, thereby inserting a marker into the target chromosome at the 5' homologous arm sequence 3', followed by insertion into the template sequence, thereby generating chromosomal rearrangements; and (c) selecting one or more cells expressing the marker. Optionally, the method includes (a) providing cells comprising a target chromosome containing a target location and a template chromosome containing a template sequence; (b) contacting the cells with a nucleic acid molecule comprising a 5' homologous arm, a marker, and a 3' homologous arm from 5' to 3', the 5' homologous arm containing a downstream nucleotide sequence from the 3' end of the template sequence, and the 3' homologous arm containing a downstream nucleotide sequence from the 3' end of the target sequence; (c) generating a double-strand break at the target location and at the 3' end of the template sequence, thereby inserting the marker into the target chromosome at the 3' of the 5' homologous arm sequence, followed by insertion into the template sequence, thereby generating a chromosomal rearrangement; and (c) selecting one or more cells expressing the marker. In some embodiments, generating a double-strand break includes contacting the cells with a CRISPR/Cas endonuclease, at least a first gNA and a second gNA, the first gNA containing a target location-specific targeting sequence such that the CRISPR/Cas endonuclease cleaves the target location, and the second gNA containing a target sequence specific to the 5' end of the template sequence. In some embodiments, generating a double-strand break includes contacting a cell with a CRISPR/Cas endonuclease, at least a first gNA and a second gNA, the first gNA containing a target-specific targeting sequence such that the CRISPR/Cas endonuclease cleaves the target site, and the second gNA containing a target sequence specific to the 3' end of a template sequence. In some embodiments, the nucleic acid molecule comprises DNA. In some embodiments, the nucleic acid molecule comprises a plasmid.

Suitable methods known in the art can be used to generate double-strand breaks in the target chromosome and the template chromosome. This can be achieved, in particular, by selecting homologous arm sequences of nucleic acid molecules (e.g., plasmids) for guiding HDR-mediated chromosomal rearrangements, said nucleic acid molecules overlapping with or containing endonuclease sites on the target chromosome and the template chromosome. In some embodiments, generating double-strand breaks in (c) includes inducing double-strand breaks using a CRISPR/Cas endonuclease and one or more guide nucleic acids (gNA), one or more zinc finger nucleases, one or more transcription activator-like effector nucleases (TALEN), or one or more CRE recombinases. For example, a Cre recombinase induces an inversion of a chromosomal region between two LoxP sites, whereby the template sequence, along with first and second markers, is inserted into the target chromosome. In some implementations, CRISPR/Cas endonucleases include CasI, CasIB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, CasX, CasY, Cas12a(Cpf1), Cas13a, CsyI, Csy2, Csy3, CseI, Cse2, CscI, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, CmrI, Cmr3, Cmr4, Cmr5, Cmr6, CsbI, Csb2, Csb3, Csx17, CsxI4, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, CsfI, Csf2, Csf3, Csf4, Cms1, C2c1, C2c2, or C2c3, or their homologs, orthologs, or modified forms. In some embodiments, the CRISPR/Cas endonuclease includes Cas9, Cas12a (Cpf1), Cas13a, CasX, CasY, C2c1, or C2c3. In some embodiments, the CRISPR/Cas endonuclease includes Cas9. In some embodiments, gNA includes a single guide RNA (sgRNA).

Any suitable method known in the art can be used to contact cells with the endonucleases described herein. For example, nucleic acid molecules (e.g., plasmids, etc.) containing the endonuclease and a sequence encoding gRNA (for CRISPR/Cas endonucleases) can be used to transfect cells. Alternatively, the endonuclease or a nucleic acid molecule encoding the endonuclease can be introduced into cells via electroporation, lipid transfection, transduction, etc.

The cells used to implement the methods described herein can be any suitable cells known in the art. In some embodiments, the cells comprise embryonic stem (ES) cells. In some embodiments, the cells comprise embryonic hybrid stem (EHS) stem cells. EHS cells can be generated by fusing ES cells from two different species (e.g., human and mouse, human and rat, or mouse and monkey). All fusion methods known in the art are contemplated within the scope of this disclosure, including but not limited to electrofusion, viral-induced fusion, and chemically induced fusion. In some embodiments, the method comprises fusing human EH cells with EH cells selected from the group consisting of: mice, rats, rabbits, guinea pigs, hamsters, sheep, goats, donkeys, cattle, horses, camels, chickens, and monkeys. In some embodiments, the method includes fusing EH cells from any two different species selected from the group consisting of: mice, rats, rabbits, guinea pigs, hamsters, sheep, goats, donkeys, cattle, horses, camels, chickens, and monkeys.

In some implementations, the cells include zygotes. As used herein, the term "zygote" refers to a eukaryotic cell formed by a fertilization event between two gametes (e.g., an ovum and a sperm in a mammal). Single-cell, 2-cell, 4-cell, 8-cell, or more advanced stages of zygotes may be suitable for the methods described herein.

Once engineered chromosomes are generated as described herein, they can be recovered using any suitable method. In some embodiments, the recovery of engineered chromosomes disclosed herein includes microcell-mediated chromosome transfer (MMCT). The recovered chromosomes are transferred to any cell type suitable for downstream applications by fusing micronucleated cells containing engineered chromosomes with target cells such as ES cells. These methods are described in more detail below.

template chromosome

This disclosure provides a template chromosome containing a template sequence for use in the methods described herein.

As used herein, "template chromosome" refers to a chromosome containing a "template sequence". A template sequence is a sequence introduced into a target chromosome or target location using the methods of this disclosure.

The template chromosome can be isolated or obtained from any suitable source. In some embodiments, the template chromosome is derived from a eukaryote. In some embodiments, the eukaryote is a vertebrate, such as a bird, reptile, or mammal. In some embodiments, the template chromosome is derived from a mouse, rat, rabbit, guinea pig, hamster, sheep, goat, donkey, cow, horse, camel, monkey, or chicken. In some embodiments, the template chromosome is derived from a human.

In some implementations, the template chromosome is a foreign chromosome, and the template sequence is a foreign sequence. For example, the target chromosome is a mouse chromosome, and the template chromosome and the corresponding template sequence are derived from a non-mouse species, such as humans.

In some implementations, the template chromosome is an endogenous chromosome, and the template sequence is an endogenous sequence. For example, the template chromosome is a mouse chromosome, while the target chromosome is a second, different mouse chromosome.

In some implementations, the template chromosome is an artificial chromosome.

In some implementations, the template chromosome is a naturally occurring chromosome.

In some implementations, the template chromosome contains one or more modifications to a naturally occurring chromosome. Modifications include, in particular, sequence insertions, deletions, and rearrangements. Examples of sequences inserted into the template chromosome include, in particular, markers, promoters, cDNA sequences, non-coding sequences, etc.

In some embodiments, the template chromosome contains an endonuclease site located at the 5' of the template sequence. In some embodiments, the template chromosome contains an endonuclease site located at the 3' of the template sequence. In some embodiments, the endonuclease site is adjacent to the template sequence. In some embodiments, the endonuclease site is located near the template sequence.

In some embodiments, the template chromosome contains endonuclease sites on either side of the template sequence. For example, the template chromosome contains a first endonuclease site at the 5' of the template sequence and a second endonuclease site at the 3' of the template sequence. In some embodiments, both the first and second endonuclease sites are recognized and cleaved by the same endonuclease. For example, both the first and second endonuclease sites contain the same DNA sequence, which is recognized by the same endonuclease. In some embodiments, the first endonuclease site is cleaved by a first endonuclease, and the second endonuclease site is cleaved by a second endonuclease. For example, the first and second endonuclease sites contain different DNA sequences recognized by two different zinc finger nucleases (ZFNs), or two different CRISPR/Cas target sequences recognized by a CRISPR/Cas ribonucleoprotein complex containing guide nucleic acids (gNAs) with different target sequences. In some embodiments, the first and/or second endonuclease sites are adjacent to the template sequence. In some embodiments, the first and/or second endonuclease sites are located near the template sequence.

Sequences within 5 base pairs (bp) of the template sequence, or within 10 bp, 15 bp, 20 bp, 30 bp, 40 bp, 50 bp, 70 bp, 80 bp, 90 bp, 100 bp, 120 bp, 140 bp, 160 bp, 180 bp, 200 bp, 250 bp, 300 bp, 400 bp, or 500 bp, can be considered close to the template sequence.

In some embodiments, the template chromosome includes one or more sequences of homologous arms of nucleic acid molecules for promoting homology-directed repair. In some embodiments, the template chromosome includes homologous arm sequences located at or near the 5' end of the template sequence. In some embodiments, the homologous arm is located upstream of the template sequence, i.e., at the 5' end of the template sequence. In some embodiments, the template chromosome includes a restriction endonuclease site, homologous arm sequences, and the template sequence from 5' to 3'. In some embodiments, the template chromosome includes homologous arm sequences located at or near the 3' end of the template sequence or at the 5' end of the template sequence. In some embodiments, the homologous arm is located downstream of the template sequence, i.e., at the 3' end of the template sequence. In some embodiments, the template chromosome includes the template sequence, homologous arm sequences, and a restriction endonuclease site from 5' to 3'. In some embodiments, the homologous arm sequences are located between the restriction endonuclease site and the template sequence.

In some embodiments, the template chromosome includes a first homologous arm sequence located at or near the 5' end of the template sequence and a second homologous arm sequence located at or near the 3' end of the template sequence; that is, the template chromosome includes homologous arms upstream and downstream of the template sequence. In some embodiments, the first homologous arm is the 3' homologous arm of a first nucleic acid molecule, which, from 5' to 3', includes a 5' homologous arm containing a nucleotide sequence upstream of the 5' end of the target sequence, at least a first labeled sequence, and the first homologous arm sequence. In some embodiments, the second homologous arm is the 5' homologous arm of a second nucleic acid molecule, which, from 5' to 3', includes a second homologous arm sequence, at least a second labeled sequence, and a 3' homologous arm containing a nucleotide sequence downstream of the 3' end of the target sequence. In some embodiments, the template chromosome, from 5' to 3', includes a first endonuclease site, a first homologous arm sequence, a template sequence, a second homologous arm sequence, and a second endonuclease site.

In some embodiments, the first and/or second homologous arm sequences are adjacent to the first and/or second endonuclease sites. In some embodiments, the first homologous arm sequence is adjacent to the first endonuclease site, and the second homologous arm sequence is adjacent to the second endonuclease site, wherein the first homologous arm is located between the first endonuclease site and the template sequence, and the second homologous arm is located between the template sequence and the second template sequence. In some embodiments, the first homologous arm is located between the first endonuclease site and the template sequence, and the second homologous arm is located between the template sequence and the second template sequence.

In some implementations, the first and/or second homologous arm sequences are located near the template sequence. Homologous arms within 0 bp, 5 base pairs (bp), 10 bp, 15 bp, 20 bp, 30 bp, 40 bp, 50 bp, 70 bp, 80 bp, 90 bp, 100 bp, 120 bp, 140 bp, 160 bp, 180 bp, 200 bp, or 250 bp of the template sequence can be considered close to the template sequence.

In some implementations, the template chromosome from 5' to 3' includes a first endonuclease site, a first homologous arm, a template sequence, a second homologous arm, and a second endonuclease site.

In some embodiments, the length of the first and/or second homologous sequences of the template chromosome is between about 20 bp and 2,000 bp, between about 50 bp and 1,500 bp, between about 100 bp and 1,400 bp, between about 150 bp and 1,300 bp, between about 200 bp and 1,200 bp, between about 300 bp and 1,100 bp, between about 400 bp and 1,000 bp, or between about 500 bp and 900 bp, or between about 600 bp and 1,200 bp. In some embodiments, the length of the homologous sequences of the template chromosome is between about 400 bp and 1,500 bp. In some embodiments, the length of the homologous sequences of the template chromosome is between about 500 bp and 1,300 bp. In some embodiments, the length of the homologous sequences of the template chromosome is between about 600 bp and 1,000 bp.

template sequence

The template chromosome contains the template sequence and serves as the source of the template sequence in the engineered chromosomes and methods described herein. The template sequence can be located at any suitable location on the template chromosome. For example, if it is not desired to be bound by theory, the template sequence can be located in a region on the template chromosome characterized by euchromatin.

The template sequence can be isolated or derived from any suitable source. In some embodiments, the template sequence comprises an endogenous sequence, such as a sequence that is endogenous to the template chromosome or to the species that produces the target chromosome. In some embodiments, the template sequence is an exogenous sequence. For example, the template sequence is derived from a sequence that is exogenous to the species that produces the target chromosome. In some embodiments, the template sequence comprises a naturally occurring sequence. In some embodiments, the template sequence comprises one or more modifications to the naturally occurring sequence. Modifications include, in particular, the insertion, deletion, and rearrangement of sequences such as artificial sequences or markers. In some embodiments, the template sequence comprises an artificial sequence. In some embodiments, the template sequence comprises both naturally occurring and artificial sequences. Exemplary artificial sequences include, in particular, markers, cDNA sequences, promoters, and recombinant sequences. Exemplary markers include, but are not limited to, the selection markers disclosed in Table 3 below, as well as detectable markers such as green fluorescent protein (GFP), mCherry, etc.

In some embodiments, the template sequence is derived from a eukaryote. In some embodiments, the eukaryote is a vertebrate, such as a bird, reptile, or mammal. In some embodiments, the template sequence comprises a mouse, rat, rabbit, guinea pig, hamster, sheep, goat, donkey, cow, horse, camel, monkey, or chicken sequence. In some embodiments, the template sequence comprises a human sequence.

In some implementations, the template sequence length is at least 25KB, at least 50KB, at least 100KB, at least 200KB, at least 400KB, at least 500KB, at least 600KB, at least 700KB, at least 800KB, at least 900KB, at least 1MB, at least 2MB, at least 3MB, at least 4MB, at least 5MB, at least 6MB, at least 7MB, at least 8MB, at least 9MB, at least 10MB, at least 15MB, at least 20MB, at least 25MB, at least 30MB, at least 40MB, at least 50MB, at least 60MB, at least 70MB, at least 80MB, at least 90MB, at least 100MB, at least 120MB, at least 140MB, at least 160MB, at least 180MB, at least 200MB, at least 220MB, or at least 250MB. In some embodiments, the template sequence length is at least 50KB, at least 100KB, at least 200KB, at least 500KB, at least 700KB, at least 1MB, at least 2MB, at least 3MB, at least 4MB, at least 5MB, at least 6MB, at least 7MB, at least 8MB, at least 9MB, at least 10MB, at least 20MB, at least 30MB, at least 40MB, or at least 50MB. In some embodiments, the template sequence length is at least 1MB. In some embodiments, the template sequence length is at least 2MB. In some embodiments, the template sequence length is at least 3MB. In some embodiments, the template sequence length is at least 4MB. In some embodiments, the template sequence length is at least 5MB. In some embodiments, the template sequence length is at least 10MB. In some embodiments, the template sequence length is at least 20MB.

In some implementations, the template sequence length is between 50KB and 250MB, between 50KB and 100MB, between 50KB and 50MB, between 50KB and 20MB, between 50KB and 10MB, between 50KB and 5MB, between 50KB and 3MB, between 50KB and 2MB, between 50KB and 1MB, between 100KB and 200MB, between 100KB and 100MB, between 100KB and 50MB, between 100KB and 200MB. Between MB, between 100KB and 10MB, between 100KB and 5MB, between 100KB and 3MB, between 100KB and 2MB, between 100KB and 1MB, between 100KB and 500KB, between 200KB and 100MB, between 200KB and 50MB, between 200KB and 20MB, between 200KB and 10MB, between 200KB and 5MB, between 200KB and 3MB, between 200KB and 2MB, between... Between 200KB and 1MB, Between 200KB and 500KB, Between 500KB and 100MB, Between 500KB and 50MB, Between 500KB and 20MB, Between 500KB and 10MB, Between 500KB and 5MB, Between 500KB and 3MB, Between 500KB and 2MB, Between 500KB and 1MB, Between 1MB and 100MB, Between 1MB and 50MB, Between 1MB and 20MB, Between 1MB and 10MB The template sequence length is between 1MB and 5MB, between 1MB and 3MB, between 1MB and 2MB, between 3MB and 100MB, between 3MB and 50MB, between 3MB and 20MB, between 3MB and 10MB, between 3MB and 5MB, between 5MB and 100MB, between 5MB and 50MB, between 5MB and 20MB, between 5MB and 10MB, between 10MB and 100MB, between 10MB and 50MB, or between 10MB and 20MB. In some embodiments, the template sequence length is between 50KB and 250MB. In some embodiments, the template sequence length is between 500KB and 200MB. In some embodiments, the template sequence length is between 200KB and 50MB, between 1MB and 20MB, between 1MB and 10MB, between 1MB and 5MB, between 1MB and 3MB, between 3MB and 20MB, between 3MB and 10MB, between 3MB and 7MB, or between 3MB and 5MB. In some embodiments, the template sequence length is between 1MB and 10MB. In some embodiments, the template sequence length is between 1MB and 5MB. In some embodiments, the template sequence length is between 3MB and 5MB.

In some embodiments, the template sequence comprises the sequences of one or more genes. In some embodiments, the template sequence comprises the sequences of multiple genes. In some embodiments, the template sequence comprises the sequences of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 genes.

In some embodiments, the template sequence comprises a human sequence, such as the sequence of one or more human genes. In some embodiments, the template sequence comprises a subsequence of a human gene. In some embodiments, the template sequence comprises a subsequence of a human gene and an artificial sequence, such as a marker or fusion protein. In some embodiments, the template sequence comprises the sequence of one or more human genes and an artificial sequence.

In some implementations, the template sequence contains a human gene sequence. It is envisioned that all human genes are within the scope of this disclosure. Without being bound by theory, transferring human genes involved in disease pathogenesis or serving as potential therapeutic targets into model organisms such as mice can facilitate disease research and the development of appropriate therapies.

Exemplary genes included in the template sequence include, but are not limited to, immunoglobulin genes, T-cell receptor (TCR) genes, immune checkpoint genes, cytokines, chemokines, receptors, transcription factors, cytoskeleton genes, cell cycle check genes, oncogenes, and genes related to development, immunology, or neurobiology. Exemplary immune checkpoint genes include BTLA, CTLA-4, TIM-3, PD-1, and PD-L1. Exemplary cytokines include interleukins (CTNF, IL-16, IL-1B, IL-6, IL-12, IL-17F, IL-2, IL-3, IL-9, IL-12B, IL-18BP, IL-21, IL-33, leptin, IL-13, IL-1A, IL-23, IL-4), interferons (IFN10, IFN-α7, IFNa4Fc, IFNβ, IFNα4, IFNγ, IFNα5, IFNω), and tumor necrosis factors (TNFs, such as BAFF, TNFβ, CD30 ligand, TNFα, CD40 ligand, TNFSF10, CD27 ligand). Exemplary chemokines include CXC, CC, CX3C, and C family chemokines. Exemplary receptors include G protein-coupled receptors, ligand-gated ion channels (ionotropic receptors), kinase-linked receptors and related receptors, and nuclear receptors. Exemplary transcription factors include, but are not limited to, helix-turn-helix transcription factors (e.g., Oct-1), helix-loop-helix transcription factors (e.g., E2A), zinc finger transcription factors (e.g., glucocorticoid receptor, GATA protein), basic protein-leucine zipper transcription factors (e.g., cyclic AMP response element binding factor (CREB) and activator protein-1 (AP-1)), and β-sheet motif transcription factors (e.g., nuclear factor-κB (NF-κB)). Exemplary cell cycle regulatory genes include, but are not limited to, cyclins, cyclin-dependent kinases, and cell cycle checkpoint genes.

In some implementations, the template sequence contains an oncogene or a tumor suppressor gene. Exemplary oncogenes and tumor suppressor genes suitable for inclusion in the template sequence are listed in Table 1 below.

Table 1. Oncogenes and tumor suppressor factors

In some embodiments, the template sequence comprises sequences of human genes associated with a genetic disease or condition. In some embodiments, the template sequence comprises sequences of human chromosomal regions associated with a genetic disease or condition. Non-limiting examples of genes and chromosomal regions associated with diseases or conditions are shown in Table 2 below.

Table 2. Genetic diseases or conditions, and related genes or genomic regions.

In some embodiments, the template sequence comprises an immunoglobulin sequence. Both surface immunoglobulins and secreted immunoglobulins are considered to be within the scope of this invention. Immunoglobulins recognize foreign antigens and initiate an immune response. In humans, each immunoglobulin molecule consists of two identical heavy chains and two identical light chains, the heavy chains being encoded by the IGH locus on chromosome 14, and the light chains being encoded by the immunoglobulin κ locus (IGK) on chromosome 2 and the immunoglobulin λ locus (IGL) on chromosome 22. The IGH locus includes a V (variable) region, a D (diversity) region, a J (connection) region, and a C (constant) region. Regions V, D, and J each contain multiple distinct gene segments, collectively referred to herein as the IGH variable region. During B cell development, a DNA-level recombination event connects a single D segment to a J segment; then, the fused DJ exon of this partially rearranged DJ region is connected to the V segment. The rearranged VDJ region, containing the fused VDJ exon, is then transcribed and fused to the constant region via RNA splicing. This transcript encodes the μ heavy chain. During late development, B cells produce VDJ-Cμ-Cδ premessenger RNA, which is selectively spliced to encode either the μ or δ heavy chain. Mature B cells in lymph nodes undergo switch recombination, resulting in a fused VDJ gene segment that approximates one of the IGHG, IGHA, or IGHE gene segments, with each cell expressing either the γ, α, or ε heavy chain. The potential recombination of many different V segments with several J segments provides broad antigen recognition. Additional diversity is achieved through linker diversity, which arises from the random addition of nucleotides by terminal deoxyribonucleoside transferases and somatic hypermutation. Each light chain consists of two tandem immunoglobulin domains: a constant domain ( _CL1 ) and a variable domain ( _V2 ). For light chains, the V domain is encoded by two separate DNA segments. The first segment is called the V gene segment because it encodes the majority of the V domain. The second segment encodes the remainder of the V domain and is referred to as the linker or J gene segment. Like the heavy chain, the light chain rearranges to link the V segment to the J gene segment and bring the V gene closer to the constant region sequence, then separates only by introns. IGH sequences of any one of IGHV, IGHD, IGHJ, IGHG, or IGHA, or any combination thereof, are considered within the scope of the template sequences disclosed herein. Light chain sequences of IGK or IGL, or combinations thereof, are considered within the scope of the template sequences disclosed herein.

In some embodiments, the engineered chromosome includes a mouse chromosome in which one or more non-coding sequences may have been introduced. For example, one or more non-coding sequences capable of regulating antibody production, maturation, and/or diversification may have been introduced into the chromosome. For example, one or more non-coding sequences capable of regulating antibody diversification may have been introduced into the chromosome. For example, one or more non-coding sequences capable of regulating antibody class switching may have been introduced into the chromosome. For example, one or more non-coding sequences within a switching region may have been introduced into the chromosome. For example, when one or more non-coding sequences have been introduced into the chromosome, class switching recombination, somatic hypermutation, and/or activation-induced cytidine deaminase can be regulated. For example, when one or more non-coding sequences have been introduced into the chromosome, the diversity of the Ig sequence library can be regulated. For example, approximately 2 kb of variable regions containing rearranged genes at the heavy chain, κ light chain, and λ light chain loci, and/or approximately 4 kb of switching regions containing a large number of G:C-rich DNA segments at the heavy chain loci, may have been introduced into the chromosome.

In some embodiments, the template sequence comprises a human IGH sequence. Human IGH spans nucleotide positions 105,586,437 to 106,879,844 on chromosome 14 of the GRCh38.p13 assembly of the human genome. Those skilled in the art will understand that a human IGH sequence with 5' and 3' boundaries is a suitable template sequence, said boundaries deviating from those described above by, for example, at least 100 bp, 500 bp, 1,000 bp, 2,000 bp, 5,000 bp, 10,000 bp, or more.

In some embodiments, the template sequence comprises a human IGH variable region sequence. In some embodiments, the human IGH variable region sequence comprises sequences encoding human _VH , _DH , and _JH 1–6 gene segments and intercalated non-coding sequences. In some embodiments, the human IGH variable region sequence comprises nucleotide positions 105,862,994 to 106,811,028 on chromosome 14 of the GRCh38.p13 assembly of the human genome. In some implementations, the human IGH variable region sequence comprises nucleotide positions 105,862,994 to 106,811,028 of chromosome 14 of the GRCh38.p13 assembly of the human genome, with at least about 50 bp, 100 bp, 500 bp, 1,000 bp, 2,000 bp, 5,000 bp, 7,000 bp, 10,000 bp, 15,000 bp, 20,000 bp, or 50,000 bp removed from the 5' end, 3' end, or both ends. In some embodiments, the human IGH variable region sequence comprises nucleotide positions 105,862,994 to 106,811,028 on chromosome 14 of the GRCh38.p13 assembly of the human genome, and additional flanking sequences of at least about 50 bp, 100 bp, 500 bp, 1,000 bp, 2,000 bp, 5,000 bp, 7,000 bp, 10,000 bp, 15,000 bp, 20,000 bp, or 50,000 bp at the 5' end, 3' end, or both ends. In some embodiments, the human IGH variable region sequence comprises nucleotide positions 105,862,994 to 106,811,028 on chromosome 14 of the GRCh38.p13 assembly of the human genome, and one or more modifications thereof. Exemplary modifications include, but are not limited to, deletions (such as deletions of one or more V, D, or J segments), insertions (such as the insertion of markers), rearrangements, or combinations thereof.

In some implementations, the template sequence contains the sequence of a T cell receptor subunit (TCR). The T cell receptor (TCR) is a protein complex found on the surface of T cells or T lymphocytes,[1] which is responsible for recognizing antigen fragments as peptides that bind to major histocompatibility complex (MHC) molecules. The TCR contains a membrane-bound heterodimer protein linked by disulfide bonds, which in most cases consists of highly variable α and β chains expressed as part of a complex with invariant CD3 chain molecules (CD3δ, CD3ε, CD3γ, and CD3ζ). T cells expressing these two chains are called α:β (or αβ) T cells. A minority of T cells express alternative receptors formed by variable γ and σ chains, called γσ T cells. TCR development occurs through a lymphocyte-specific gene recombination process that assembles a large number of potential segments into a final sequence, which occurs through recombination of TCR gene segments in T cells in the thymus. The TCRα locus contains variable (V) and linker (J) gene segments (Vβ and Jβ), while the TCRβ locus contains a D gene segment in addition to the Vα and Jα segments. Therefore, the α chain is generated by VJ recombination, and the β chain participates in VDJ recombination. This is similar to the development of γδTCR, where the TCRγ chain participates in VJ recombination, and the TCRδ gene is generated by VDJ recombination. The TCRα chain locus consists of 46 variable segments, 8 linker segments, and constant regions. The TCRβ chain locus consists of 48 variable segments, followed by two diversity segments, 12 linker segments, and two constant regions. Template sequences containing any TCR subunit described herein, its subsequences, or combinations thereof are considered to be within the scope of this disclosure. In some implementations, the template sequence includes a TCRα chain variable region sequence (encoded by the T cell receptor α locus or TRA), a TCRβ chain variable region sequence (encoded by the T cell receptor β locus or TRB), a TCRγ variable region sequence (encoded by the T cell receptor γ locus or TRG), or a TCRδ variable region sequence (encoded by the T cell receptor δ locus or TRD).

In some implementations, the template sequence contains a sequence encoding an antibody or antigen-binding fragment.

As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or reacts with a specific antigen, including polyclonal antibodies, monoclonal antibodies, genetically engineered antibodies, and other modified antibody forms, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi-, tri-, and tetra-specific antibodies, double-chain antibodies, triple-chain antibodies, and quadruple-chain antibodies), and antigen-binding fragments of antibodies, including, for example, Fab′, F(ab′) ₂ , Fab, Fv, rlgG, and scFv fragments. Unless otherwise stated, the term "monoclonal antibody" (mAb) means including the complete molecule as well as antibody fragments (including, for example, Fab and F(ab′) ₂ fragments) capable of specifically binding to a target protein. As used herein, Fab and F(ab′) ₂ fragments refer to antibody fragments lacking the Fc fragment of the complete antibody. Examples of such antibody fragments are described herein.

As used herein, the term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be achieved through fragments of a full-length antibody. Antibody fragments can be, for example, Fab, F(ab′) ₂ , scFv, double-stranded antibodies, triple-stranded antibodies, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab′)2 fragments, containing a divalent fragment containing two Fab fragments linked by disulfide bonds in a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of an antibody arm; (v) dAb fragments including VH and VL domains; (vi) dAb fragments consisting of VH domains (see, for example, Ward et al., Nature 341:544-546, 1989); (vii) dAbs consisting of VH or VL domains; (viii) isolated complementarity-determining regions (CDRs); and (ix) combinations of two or more (e.g., two, three, four, five, or six) isolated CDRs, which may optionally be linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be linked by a linker using recombination methods, which allows them to become a single protein chain, where the VL and VH regions pair to form a monovalent molecule (called a single-stranded Fv (scFv)); see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the applicability of the fragments can be screened in the same manner as intact antibodies. Antigen-binding fragments can be generated by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or, in some cases, by chemical peptide synthesis methods known in the art.

As used herein, the term “complementarity-determining region” (CDR) refers to a hypervariable region present in both the light and heavy chain variable domains of an antibody. The more conserved portion of the variable domain is called the frame region (FR). The amino acid positions describing the antibody’s hypervariable region can vary depending on the context and various definitions known in the art. Some positions within the variable domain can be considered hybrid hypervariable positions because they are considered within the hypervariable region under one set of criteria and outside the hypervariable region under another set of criteria. One or more of these positions can also be present in extended hypervariable regions. The antibodies described herein may contain modifications at these hybrid hypervariable positions. The variable domains of the native heavy and light chains each contain four frame regions predominantly employing a β-sheet configuration, linked by three CDRs that form loops connecting β-sheet structures and, in some cases, form part of a β-sheet structure. CDRs in each chain are tightly bound together via framework regions in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and together with CDRs from other antibody chains, contribute to the formation of the antibody's target binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md., 1987). As used herein, unless otherwise stated, immunoglobulin amino acid residues are numbered according to the immunoglobulin amino acid residue numbering system of Kabat et al.

In some embodiments, the antibody or antigen-binding fragment includes a human antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is humanized.

Those skilled in the art will understand that the template sequence may also include sequences necessary for gene expression (such as antibodies) in a particular tissue, cell type, or organism. Such sequences include, but are not limited to, promoters, enhancers, untranslated sequences such as the 5' and 3' untranslated regions of messenger RNA (mRNA), polyadenylation (polyA) sequences, introns, internal ribosome entry sites (IRES), etc. The selection of a suitable sequence will be apparent to those skilled in the art.

In some embodiments, the template sequence contains a promoter. In some embodiments, the promoter contains an endogenous promoter, i.e., a promoter that is typically associated with a gene contained in the template sequence. In some embodiments, the promoter is not an endogenous promoter, for example, a promoter isolated or derived from a gene other than the gene operatively linked to the promoter in the template sequence, or from an organism. For example, the template sequence contains a sequence encoding an antibody or antigen-binding fragment that is operatively linked to a promoter that is not an immunoglobulin promoter. In some embodiments, the promoter is a constitutive promoter, an inducible promoter, or a tissue-specific promoter. In some embodiments, the promoter is isolated from or derived from a mammalian gene, such as a gene expressed in lymphocytes.

Exemplary promoters that can be used to express the template sequence include, but are not limited to, the SV40 early promoter region, promoters contained in the 3' long terminal repeat sequence of Rous sarcoma virus, regulatory sequences of metallothionein genes, tetracycline (Tet) promoters, promoter elements from yeast or other fungi such as Gal promoters, ADC (alcohol dehydrogenase) promoters, PGK (glycerol phosphate kinase) promoters, alkaline phosphatase promoters, and the following animal transcription control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene control regions active in pancreatic acinar cells; insulin gene control regions active in pancreatic β cells; immunoglobulin gene control regions active in lymphoid cells; and regions active in testicular cells, mammary cells, lymphoid cells, and... The study included active mouse mammary tumor virus control regions in mast cells, active albumin gene control regions in the liver, active alpha-fetoprotein gene control regions in the liver, active α1-antitrypsin gene control regions in the liver, active β-globin gene control regions in myeloid cells, active myelin basic protein gene control regions in oligodendrocytes of the brain, active myosin light chain-2 gene control regions in skeletal muscle, active neuron-specific enolase (NSE) and brain-derived neurotrophic factor (BDNF) gene control regions in neuronal cells, active glial fibrillary acidic protein (GFAP) promoters in astrocytes, and active gonadotropin-releasing gene control regions in the hypothalamus.

target chromosome

This disclosure provides a target chromosome containing a target sequence for use in the methods described herein.

As used herein, "target chromosome" refers to a chromosome containing a "target sequence," or, where there is no explicit deletion of the target sequence by inserting a template sequence, a "target location." A target sequence is a target chromosome sequence deleted by inserting a template sequence using the methods described herein. A target location is the position in the target chromosome where the template sequence is inserted (for insertion) or connected to it (for chromosomal translocation or rearrangement).

The target chromosome can be isolated or derived from any suitable source. In some embodiments, the target chromosome is derived from a eukaryote. In some embodiments, the eukaryote is a vertebrate, such as a bird, reptile, or mammal. In some embodiments, the target chromosome is derived from a mouse, rat, rabbit, guinea pig, hamster, sheep, goat, donkey, cow, horse, camel, monkey, or chicken. In some embodiments, the target chromosome is derived from a mouse. In some embodiments, the target chromosome is derived from a rat. In some embodiments, the target chromosome is derived from a monkey.

In some implementations, the template chromosome and the target chromosome are from different species. For example, the template chromosome is from a human, and the target chromosome is from a mouse. In other implementations, the template chromosome and the target chromosome are from the same species.

In some implementations, the target chromosome is an artificial chromosome.

In some implementations, the target chromosome is a naturally occurring chromosome.

In some implementations, the target chromosome contains one or more modifications to a naturally occurring chromosome. Modifications include, in particular, sequence insertions, deletions, and rearrangements. Examples of sequences inserted into the target chromosome include, in particular, markers, promoters, cDNA sequences, non-coding sequences, etc. Suitable markers include selection markers, such as those disclosed in Table 3, and detectable markers, such as GFP, mCherry, etc.

In some embodiments, the target chromosome contains an endonuclease site located at the 5' of the template sequence. In some embodiments, the target chromosome contains an endonuclease site located at the 3' of the target sequence. In some embodiments, the endonuclease site is adjacent to the target sequence. In some embodiments, the endonuclease site is located near the target sequence.

In some embodiments, the target chromosome contains endonuclease sites on either side of the target sequence. For example, the target chromosome contains a first endonuclease site at the 5' of the target sequence and a second endonuclease site at the 3' of the target sequence. In some embodiments, both the first and second endonuclease sites are recognized and cleaved by the same endonuclease. For example, both the first and second endonuclease sites contain the same DNA sequence, which is recognized by the same endonuclease. In some embodiments, the first endonuclease site is cleaved by a first endonuclease, and the second endonuclease site is cleaved by a second endonuclease. For example, the first and second endonuclease sites contain different DNA sequences recognized by two different zinc finger nucleases (ZFNs), or two different CRISPR/Cas target sequences recognized by a CRISPR/Cas ribonucleoprotein complex containing guide nucleic acids (gNAs) with different target sequences. In some embodiments, the first and/or second endonuclease sites are adjacent to the target sequence. In some embodiments, the first and/or second endonuclease sites are located near the target sequence.

Endonuclease sites within 5 base pairs (bp), 10bp, 15bp, 20bp, 30bp, 40bp, 50bp, 70bp, 80bp, 90bp, 100bp, 120bp, 140bp, 160bp, 180bp, 200bp, 250bp, 300bp, 400bp, or 500bp of the template sequence are considered to be close to the target sequence.

In some embodiments, the target chromosome includes one or more sequences of homologous arms of nucleic acid molecules for promoting homology-directed repair. In some embodiments, the target chromosome includes a homologous arm sequence located at the 5' of the target sequence. In some embodiments, the target chromosome includes a homologous arm sequence, a nuclease site, and the target sequence from 5' to 3'. In some embodiments, the target chromosome includes a homologous arm sequence located at the 3' of the target sequence. In some embodiments, the target chromosome includes the target sequence, a nuclease site, and a homologous arm sequence from 5' to 3'. In some embodiments, the nuclease site is located between the homologous arm sequence and the target sequence.

In some embodiments, the target chromosome comprises a 5' first homologous arm sequence and a 3' second homologous arm sequence of the target sequence. That is, the target chromosome contains homologous arms both upstream and downstream of the target sequence. In some embodiments, the first homologous arm is the 5' homologous arm of a first nucleic acid molecule, which comprises, from 5' to 3', a first homologous arm, at least a first labeled sequence, and a 3' homologous arm containing a nucleotide sequence upstream of the 5' end of the template sequence. In some embodiments, the second homologous arm is the 3' homologous arm of a second nucleic acid molecule, which comprises, from 5' to 3', a 5' homologous arm containing a nucleotide sequence downstream of the 3' end of the template sequence, at least a second labeled sequence, and a second homologous arm. In some embodiments, the target chromosome comprises, from 5' to 3', a first homologous arm sequence, a first endonuclease site, the target sequence, a second endonuclease site, and a second homologous arm sequence.

In some embodiments, the first and/or second homologous arm sequence of the target chromosome is adjacent to the first and/or second endonuclease site. In some embodiments, the first homologous arm sequence is adjacent to the first endonuclease site, and the second homologous arm sequence is adjacent to the second endonuclease site, wherein the first endonuclease site is located between the first homologous arm and the target sequence, and the second endonuclease site is located between the target sequence and the second homologous arm.

In some implementations, the first and/or second homologous arm sequences are located near the target sequence. Endonuclease sites located within 5 bp, 10 bp, 15 bp, 20 bp, 30 bp, 40 bp, 50 bp, 70 bp, 80 bp, 90 bp, 100 bp, 120 bp, 140 bp, 160 bp, 180 bp, 200 bp, or 250 bp of the target sequence can be considered close to the target sequence.

In some implementations, the target chromosome from 5' to 3' includes a first homologous arm, a first endonuclease site, a target sequence, a second endonuclease site, and a second homologous arm.

In some embodiments, when the template sequence is inserted, little or no target chromosome sequence is deleted, and the target sequence is interchangeably referred to herein as a "target site" or "target location." Those skilled in the art will understand that in these cases, the arrangement of homologous arms and endonuclease sites is similar to those described above, except that the homologous arms at the target location are flanked by the endonuclease site, rather than flanking the target sequence itself. In some embodiments, the target chromosome from 5' to 3' comprises the sequence of a first homologous arm, the endonuclease site, and the sequence of a second homologous arm. In some embodiments, the first homologous arm is the 5' homologous arm of a first nucleic acid molecule, which from 5' to 3' comprises the first homologous arm, at least a first labeled sequence, and a 3' homologous arm comprising a nucleotide sequence upstream of the 5' end of the template sequence. In some embodiments, the second homologous arm is the 3' homologous arm of a second nucleic acid molecule, which from 5' to 3' comprises a 5' homologous arm containing a nucleotide sequence downstream of the 3' end of the template sequence, at least a second labeled sequence, and a second homologous arm.

In some embodiments, the connection between the template sequence and the target sequence results in chromosomal rearrangements or translocations. In some embodiments, the target chromosome includes a target chromosome homologous arm sequence and a nuclease site from 5' to 3'. In some embodiments, the target chromosome homologous arm includes a 5' homologous arm of a nucleic acid molecule, said nucleic acid molecule including a target sequence homologous arm from 5' to 3', at least one marker, and a 3' homologous arm containing a nucleotide sequence upstream of the 5' end of the template sequence. In some embodiments, the target chromosome includes a nuclease site and a target chromosome homologous arm sequence from 5' to 3'. In some embodiments, the target chromosome homologous arm includes a 3' homologous arm of a nucleic acid molecule, said nucleic acid molecule including a 5' homologous arm containing a nucleotide sequence downstream of the 3' end of the template sequence, at least a first marker, and a target sequence homologous arm.

In some embodiments, the length of the first and/or second homologous arm sequence of the target chromosome is between about 20 bp and 2,000 bp, between about 50 bp and 1,500 bp, between about 100 bp and 1,400 bp, between about 150 bp and 1,300 bp, between about 200 bp and 1,200 bp, between about 300 bp and 1,100 bp, between about 400 bp and 1,000 bp, or between about 500 bp and 900 bp, or between about 600 bp and 800 bp. In some embodiments, the length of the homologous sequence of the target chromosome is between about 400 bp and 1,500 bp. In some embodiments, the length of the homologous sequence of the target chromosome is between about 500 bp and 1,300 bp. In some embodiments, the length of the homologous sequence of the target chromosome is between about 600 bp and 1,000 bp.

Target sequence or target location

The target chromosome contains a target sequence or location in which the template sequence is inserted, or a target sequence or location to which the template sequence is linked by the methods described herein. The target sequence may be located at any suitable location on the target chromosome.

The target sequence can be isolated or derived from any suitable source. In some embodiments, the target sequence and the template sequence are from different species. For example, the template sequence is from a human, while the target sequence is from a mouse. In some embodiments, the target sequence and the template sequence are from the same species.

In some embodiments, the target sequence includes a naturally occurring sequence. In some embodiments, the target sequence includes one or more modifications to the naturally occurring sequence. Modifications include, in particular, the insertion, deletion, and rearrangement of sequences such as artificial sequences or markers. In some embodiments, the target sequence includes an artificial sequence. In some embodiments, the target sequence includes both naturally occurring and artificial sequences. Exemplary artificial sequences include, in particular, markers, cDNA sequences, promoters, and recombinant sequences. Exemplary markers include, but are not limited to, the selection markers disclosed in Table 3 below, as well as detectable markers such as green fluorescent protein (GFP), mCherry, etc.

In some embodiments, the target sequence is derived from a eukaryote. In some embodiments, the eukaryote is a vertebrate, such as a bird, reptile, or mammal. In some embodiments, the template sequence comprises a mouse, rat, rabbit, guinea pig, hamster, sheep, goat, donkey, cow, horse, camel, monkey, or chicken sequence. In some embodiments, the target sequence comprises a mouse sequence. In some embodiments, the target sequence comprises a rat sequence. In some embodiments, the target sequence comprises a monkey sequence.

In some implementations, the target sequence length is at least 25KB, at least 50KB, at least 100KB, at least 200KB, at least 400KB, at least 500KB, at least 600KB, at least 700KB, at least 800KB, at least 900KB, at least 1MB, at least 2MB, at least 3MB, at least 4MB, at least 5MB, at least 6MB, at least 7MB, at least 8MB, at least 9MB, at least 10MB, at least 15MB, at least 20MB, at least 25MB, at least 30MB, at least 40MB, at least 50MB, at least 60MB, at least 70MB, at least 80MB, at least 90MB, at least 100MB, at least 120MB, at least 140MB, at least 160MB, at least 180MB, at least 200MB, at least 220MB, or at least 250MB. In some embodiments, the target sequence length is at least 50KB, at least 100KB, at least 200KB, at least 500KB, at least 700KB, at least 1MB, at least 2MB, at least 3MB, at least 4MB, at least 5MB, at least 6MB, at least 7MB, at least 8MB, at least 9MB, at least 10MB, at least 20MB, at least 30MB, at least 40MB, or at least 50MB. In some embodiments, the target sequence length is at least 1MB. In some embodiments, the target sequence length is at least 2MB. In some embodiments, the target sequence length is at least 3MB. In some embodiments, the target sequence length is at least 4MB. In some embodiments, the target sequence length is at least 5MB. In some embodiments, the target sequence length is at least 10MB. In some embodiments, the target sequence length is at least 20MB.

In some implementations, the target sequence length is between 50KB and 250MB, between 50KB and 100MB, between 50KB and 50MB, between 50KB and 20MB, between 50KB and 10MB, between 50KB and 5MB, between 50KB and 3MB, between 50KB and 2MB, between 50KB and 1MB, between 100KB and 200MB, between 100KB and 100MB, between 100KB and 50MB, between 100KB and 20MB. Between B, between 100KB and 10MB, between 100KB and 5MB, between 100KB and 3MB, between 100KB and 2MB, between 100KB and 1MB, between 100KB and 500KB, between 200KB and 100MB, between 200KB and 50MB, between 200KB and 20MB, between 200KB and 10MB, between 200KB and 5MB, between 200KB and 3MB, between 200KB and 2MB, between 2 Between 0KB and 1MB, between 200KB and 500KB, between 500KB and 100MB, between 500KB and 50MB, between 500KB and 20MB, between 500KB and 10MB, between 500KB and 5MB, between 500KB and 3MB, between 500KB and 2MB, between 500KB and 1MB, between 1MB and 100MB, between 1MB and 50MB, between 1MB and 20MB, between 1MB and 10MB. Between 1MB and 5MB, between 1MB and 3MB, between 1MB and 2MB, between 3MB and 100MB, between 3MB and 50MB, between 3MB and 20MB, between 3MB and 10MB, between 3MB and 5MB, between 5MB and 100MB, between 5MB and 50MB, between 5MB and 20MB, between 5MB and 10MB, between 10MB and 100MB, between 10MB and 50MB, or between 10MB and 20MB. In some embodiments, the target sequence length is between 200KB and 50MB, between 1MB and 20MB, between 1MB and 10MB, between 1MB and 5MB, between 1MB and 3MB, between 3MB and 20MB, between 3MB and 10Mb, between 3MB and 7MB, or between 3MB and 5MB. In some embodiments, the target sequence length is between 1MB and 10MB. In some implementations, the target sequence length is between 1 MB and 5 MB. In some implementations, the target sequence length is between 3 MB and 5 MB.

In some embodiments, the target sequence comprises the sequences of one or more genes. In some embodiments, the target sequence comprises the sequences of multiple genes. In some embodiments, the target sequence comprises the sequences of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 genes.

In some implementations, the target sequence comprises a sequence homologous to the template sequence. For example, the template chromosome is a human chromosome containing a human template sequence comprising one or more genes described in Tables 1 and 2 above, while the target chromosome is a mouse chromosome containing a mouse target sequence, and the mouse target sequence comprises a mouse sequence homologous to the human template sequence. As another example, the template chromosome is a human chromosome containing a human IGH sequence, while the target chromosome is a mouse chromosome, and the target sequence comprises a homologous mouse Igh sequence. As yet another example, the template chromosome is a human chromosome containing a human TCR sequence, while the target chromosome is a mouse chromosome, and the target sequence comprises a homologous mouse TCR sequence.

In some implementations, the target chromosome is derived from a mouse, rat, rabbit, guinea pig, hamster, sheep, goat, donkey, cow, horse, camel, monkey, or chicken, and the target sequence contains a homolog of the template sequence from the mouse, rat, rabbit, guinea pig, hamster, sheep, goat, donkey, cow, horse, camel, monkey, or chicken.

In some embodiments, the target sequence comprises a sequence of a gene from a mouse, rat, rabbit, guinea pig, hamster, sheep, goat, donkey, cow, horse, camel, monkey, or chicken. All genes from mice, rats, rabbits, guinea pigs, hamsters, sheep, goats, donkeys, cows, horses, camels, monkeys, or chickens are considered to be within the scope of this disclosure. It is not desirable to be bound by theory; transferring human genes involved in disease pathogenesis or serving as potential therapeutic targets into model organisms such as mice, rats, rabbits, guinea pigs, hamsters, sheep, goats, donkeys, cows, horses, camels, monkeys, or chickens can facilitate disease research and the development of appropriate therapies. In some embodiments, the target sequence comprises a mouse sequence homologous to a human template sequence. In some embodiments, the target sequence comprises a rat sequence homologous to a human template sequence. In some embodiments, the target sequence comprises a monkey sequence homologous to a human template sequence.

In some embodiments, the target sequence comprises an immunoglobulin sequence, such as a mouse immunoglobulin sequence. In some embodiments, the target sequence comprises a mouse Igh sequence. The mouse Igh sequence spans nucleotide positions 1112,947,269 to 116,248,693 on chromosome 12 of the GRCm39 assembly of the mouse genome. Those skilled in the art will understand that a mouse Igh sequence with 5' and 3' boundaries is a suitable template sequence, said boundaries deviating from those described above, for example, by at least 100 bp, 500 bp, 1,000 bp, 2,000 bp, 5,000 bp, 10,000 bp, or more.

In some embodiments, the target sequence comprises a mouse Igh variable region sequence. In some embodiments, the mouse Igh variable region sequence comprises sequences encoding mouse homologs of the _VH , _DH , and _JH 1-6 gene segments and intercalated non-coding sequences. In some embodiments, the mouse Igh variable region sequence comprises nucleotide positions 113,391,842 to 115,973,952 on chromosome 12 of the GRCm39 assembly of the mouse genome. In some implementations, the mouse Igh variable region sequence comprises nucleotide positions 113,391,842 to 115,973,952 of chromosome 12 of the GRCm39 assembly of the mouse genome, with at least about 50 bp, 100 bp, 500 bp, 1,000 bp, 2,000 bp, 5,000 bp, 7,000 bp, 10,000 bp, 15,000 bp, 20,000 bp, or 50,000 bp removed from the 5' end, 3' end, or both ends. In some embodiments, the human IGH variable region sequence comprises nucleotide positions 113,391,842 to 115,973,952 on chromosome 12 of the GRCm39 assembly of the mouse genome, and additional flanking sequences of at least about 50 bp, 100 bp, 500 bp, 1,000 bp, 2,000 bp, 5,000 bp, 7,000 bp, 10,000 bp, 15,000 bp, 20,000 bp, or 50,000 bp at the 5' end, 3' end, or both ends. In some embodiments, the mouse Igh variable region sequence comprises nucleotide positions 113,391,842 to 115,973,952 on chromosome 12 of the GRCm39 assembly of the mouse genome, and one or more modifications thereof. Exemplary modifications include, but are not limited to, deletions (such as deletions of one or more V, D, or J segments), insertions (such as the insertion of markers), rearrangements, or combinations thereof. In some embodiments, the target sequence comprises a mouse Igk variable region sequence. In some embodiments, the target sequence comprises a mouse Igk variable region sequence. In some embodiments, the template sequence comprises a human IGL variable region sequence. In some embodiments, the template sequence comprises a human IGK variable region sequence.

In some embodiments (e.g., those in which the method described herein deletes little or no target chromosome sequence), the target chromosome contains a target location. A target location is either the site where the template sequence is inserted or the site where the template sequence is attached. Any location on the target chromosome can be suitable. In some embodiments, the target location contains an endonuclease site for generating a double-strand break at the target location.

engineered chromosomes

This disclosure provides engineered chromosomes produced by the methods described herein.

In some embodiments, the engineered chromosome comprises a mouse chromosome containing one or more humanized sequences. In some embodiments, the humanized sequences comprise one or more genes associated with human diseases or conditions, such as genes associated with genetic diseases or conditions, or oncogenes. In some embodiments, the engineered chromosome comprises a rat chromosome containing one or more humanized sequences. In some embodiments, the engineered chromosome comprises a monkey chromosome containing one or more humanized sequences.

In some embodiments, the engineered chromosome comprises a mouse chromosome in which one or more immunoglobulin sequences have been humanized. In some embodiments, the immunoglobulin sequences comprise IGH sequences, such as the IGH variable region. In some embodiments, the engineered chromosome comprises mouse chromosome 12, wherein the mouse Igh variable region has been replaced by a human IGH variable region derived from chromosome 14. In some embodiments, the mouse Igh variable region comprises _VH , _DH , and _JH 1-6 gene segments and intercalated non-coding sequences. In some embodiments, the human IGH variable region comprises _VH , _DH , and _JH 1-6 gene segments and intercalated non-coding sequences. In some embodiments, the engineered chromosome comprises mouse chromosome 12, wherein the mouse Igh variable region of chromosome 12, comprising nucleotide sequences from 113,391,842 to 115,973,952 of the GRCm39 assembly of the mouse genome, has been replaced by the human IGH variable region of chromosome 14, comprising nucleotide sequences from 105,862,994 to 106,811,028 of the GRCh38.p13 assembly of the human genome. In some embodiments, the engineered chromosome is mouse chromosome 6, which contains a sequence of the human IGK variable region replacing the mouse Igk variable region. In some embodiments, the mouse Igk variable region sequence comprises sequences encoding mouse _Vk and _Jk1-5 gene segments and intercalated non-coding sequences. In some embodiments, the template sequence comprises the human IGK variable region sequence. In some embodiments, the human IGK variable region sequence comprises sequences encoding human _Vk and _Jk1-5 gene segments and intercalated non-coding sequences.

Nucleic acid molecules, plasmids and vectors

This disclosure provides nucleic acid molecules for use in the methods described herein. A nucleic acid molecule, sometimes referred to as a polynucleotide, is a chain of linked nucleotides that makes up a single molecule. The nucleic acid molecules of this disclosure may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Exemplary nucleic acid molecules of the present invention contain homologous arms specific to or adjacent to the target sequence and template sequence to facilitate template sequence insertion into the target sequence or to repair double-strand breaks connecting the template and target sequences.

This disclosure provides nucleic acid molecules comprising homologous arms specific to both the target chromosome and the template chromosome, which facilitate HDR-mediated chromosomal rearrangements as described herein. In some embodiments, the nucleic acid molecule comprises a 5' homologous arm, at least a first marker, and a 3' homologous arm from 5' to 3', the 5' homologous arm containing a nucleotide sequence upstream of the 5' end of the target sequence, and the 3' homologous arm containing a nucleotide sequence upstream of the 5' end of the template sequence. In some embodiments, the nucleic acid molecule comprises a 5' homologous arm, at least a second marker, and a 3' homologous arm from 5' to 3', the 5' homologous arm containing a nucleotide sequence downstream of the 3' end of the template sequence, and the 3' homologous arm containing a nucleotide sequence downstream of the 3' end of the target sequence.

This disclosure provides vectors comprising the nucleic acid molecules described herein. According to this disclosure, a vector is a nucleic acid molecule capable of transporting other nucleic acids linked to it. For example, a plasmid is one type of vector. The vector sequence particularly includes sequences necessary for generating the vector from a host cell such as bacteria, such as origin of replication and selection markers.

In some embodiments, the vector is a plasmid. In some embodiments, the plasmid comprises a 5' homologous arm, at least a first marker, and a 3' homologous arm from 5' to 3', wherein the 5' homologous arm contains a nucleotide sequence upstream of the 5' end of the target sequence, and the 3' homologous arm contains a nucleotide sequence upstream of the 5' end of the template sequence. In some embodiments, the plasmid comprises a 5' homologous arm, at least a second marker, and a 3' homologous arm from 5' to 3', wherein the 5' homologous arm contains a nucleotide sequence downstream of the 3' end of the template sequence, and the 3' homologous arm contains a nucleotide sequence downstream of the 3' end of the target sequence.

In some embodiments, the vector includes a homologous arm sequence located at or near the 5' end of the template sequence. In some embodiments, the homologous arm is located upstream of the template sequence, i.e., at the 5' end. In some embodiments, the vector includes a homologous arm sequence located at or near the 3' end of the template sequence. In some embodiments, the homologous arm is located downstream of the template sequence, i.e., at the 3' end. In some embodiments, the sequence of the template homologous arm in the vector is identical or substantially identical to the sequence of the homologous arm in the template sequence.

In some embodiments, the vector includes a homologous arm sequence located at position 5' (i.e., upstream of the target sequence or position). In some embodiments, the vector includes a homologous arm sequence located at position 3' (i.e., downstream of the target sequence or position).

Those skilled in the art will understand that a degree of mismatch may exist between the homologous arm sequence in the vector and its equivalent sequence in the template chromosome or target chromosome, and the vector will still facilitate the repair of double-strand breaks in the template chromosome or target chromosome derived from the vector. For example, vector homologous arm sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with their equivalent sequence in the template chromosome will be suitable for the methods of this disclosure.

In some implementations, the nucleic acid molecules, plasmids, or vectors described herein contain one or more endonuclease sites.

In some embodiments, this disclosure provides (i) a first nucleic acid molecule comprising, from 5' to 3', a 5' homologous arm, at least a first label, and a 3' homologous arm, said 5' homologous arm containing a nucleotide sequence upstream of the 5' end of a target sequence, said 3' homologous arm containing a nucleotide sequence upstream of the 5' end of a template sequence; and (ii) a second nucleic acid molecule comprising, from 5' to 3', a 5' homologous arm, at least a second label, and a 3' homologous arm, said 5' homologous arm containing a nucleotide sequence downstream of the 3' end of a template sequence, said 3' homologous arm containing a nucleotide sequence downstream of the 3' end of a target sequence. In some embodiments, the first and second nucleic acid molecules are plasmids. In some embodiments, the first nucleic acid molecule comprises, from 5' to 3', a 5' homologous arm containing a nucleotide sequence upstream of the 5' end of the target sequence, a first endonuclease site, at least a first marker, a second endonuclease site, and a 3' homologous arm containing a nucleotide sequence upstream of the 5' end of the template sequence, wherein the first and second endonuclease sites overlap with the homologous arm, such that the first and second endonuclease sites on the nucleic acid molecule, as well as the corresponding endonuclease sites on the template chromosome and the target chromosome, are cleaved by the same endonuclease. In some embodiments, the second nucleic acid molecule comprises, from 5' to 3', a 5' homologous arm containing a nucleotide sequence downstream of the 3' end of the template sequence, a third endonuclease site, at least a second marker, a fourth endonuclease site, and a 3' homologous arm containing a nucleotide sequence downstream of the 3' end of the target sequence, wherein the second and third endonuclease sites overlap with the homologous arm, such that the third and fourth endonuclease sites on the nucleic acid molecule, as well as the corresponding endonuclease sites on the template chromosome and the target chromosome, are cleaved by the same endonuclease. In some embodiments, the first and second markers are not the same markers. In some embodiments, the first label on the first nucleic acid molecule includes a combination of a selectable label and a detectable label. In some embodiments, the first label includes eGFP and puromycin resistance. In some embodiments, the second label includes a selectable label. In some embodiments, the second label includes hygromycin resistance.

In some implementations, homologous arm sequences on a nucleic acid molecule correspond to sequences located near the template sequence, target sequence, or target site. Homologous arms within 0 bp, 5 base pairs (bp), 10 bp, 15 bp, 20 bp, 30 bp, 40 bp, 50 bp, 70 bp, 80 bp, 90 bp, 100 bp, 120 bp, 140 bp, 160 bp, 180 bp, 200 bp, or 250 bp of the template sequence, target sequence, or target site can be considered to be close to the said sequence.

In some embodiments, the length of the homologous sequence of the nucleic acid molecule corresponding to the template or target chromosome sequence is between about 20 bp and 2,000 bp, between about 50 bp and 1,500 bp, between about 100 bp and 1,400 bp, between about 150 bp and 1,300 bp, between about 200 bp and 1,200 bp, between about 300 bp and 1,100 bp, between about 400 bp and 1,000 bp, or between about 500 bp and 900 bp, or between about 600 bp and 800 bp. In some embodiments, the length of the homologous sequence of the nucleic acid molecule is between about 400 bp and 1,500 bp. In some embodiments, the length of the homologous sequence of the nucleic acid molecule is between about 500 bp and 1,300 bp. In some embodiments, the length of the homologous sequence of the nucleic acid molecule is between about 600 bp and 1,000 bp.

In some embodiments, the nucleic acid molecule contains a marker suitable for expression in mammalian cells. In some embodiments, the marker is located between homologous arms of the nucleic acid molecule, thereby being inserted into the target sequence. In some embodiments, the marker is a selection marker. Suitable selection markers include dihydrofolate reductase (DHFR), glutamine synthase (GS), puromycin acetyltransferase, blastcin deaminase, histidine dehydrogenase, hygromycin phosphotransferase (hph), bleomycin resistance gene, aminoglycoside phosphotransferase (neomycin resistance gene), and are further described in detail in Table 3 below.

In some embodiments, the label includes a detectable label (or reporter molecule). Detectable labels include, but are not limited to, enzymes that mediate luminescent reactions (luxA, luxB, luxAB, luc, rue, nluc), enzymes that mediate colorimetric reactions (lacZ, HRP), and fluorescent proteins such as green fluorescent protein (GFP), eGFP, yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), dsRed, mCherry, tdTomato, near-infrared fluorescent protein, etc. The selection of suitable detectable labels is known to those skilled in the art.

The tag can be expressed using any suitable promoter known in the art (including, but not limited to, the early cytomegalovirus (CMV) promoter, the PGK promoter, and the EF1a promoter).

Table 3. Selection Markers

Select tag Selecting reagents Dihydrofolate reductase (DHFR) Methionine sulfone imine (MSX) Glutamine synthase (GS) Methotrexate (MTX) Puromycin acetyltransferase Purinomycin Rice blast fungicide deaminase Rice blast fungicide Histidine dehydrogenase histidine Hygromycin phosphotransferase (hph) Hygromycin Bleomycin resistance gene bleomycin Aminoglycoside enzyme phosphotransferase Neomycin (G418)

In some embodiments (e.g., those using methods employing two nucleic acid molecules (a first nucleic acid molecule having a first label and a second nucleic acid molecule having a second label), the first or second label comprises a fluorescent protein operatively linked to a promoter capable of expressing a fluorescent protein in a cell. In some embodiments, the fluorescent protein comprises green fluorescent protein (GFP). In some embodiments, the first label further comprises a selection marker. In some embodiments, the second label further comprises a selection marker. In some embodiments, the selection marker is selected from the group consisting of dihydrofolate reductase (DHFR), glutamine synthase (GS), puromycin acetyltransferase, blastcin deaminase, histamine dehydrogenase, hygromycin phosphotransferase (hph), bleomycin resistance gene, and aminoglycoside phosphotransferase. In some embodiments, the first and second labels are not the same selection marker. In some embodiments, the first label comprises GFP operatively linked to a promoter capable of expressing GFP in a cell and puromycin acetyltransferase, and the second label comprises hygromycin phosphotransferase.

Methods to induce double-strand breaks

This article presents a method for generating double-strand breaks in template and target chromosomes. The method described herein utilizes repair pathways designed for double-strand break repair in the cellular environment to facilitate the transfer of large sequences between chromosomes.

Any method known in the art for generating double-strand breaks in DNA sequences, and any repair pathway for repairing such double-strand breaks, is considered to be within the scope of this disclosure.

In some embodiments, double-strand breaks in the template and target chromosomes are generated using one or more endonucleases. In some embodiments, the endonucleases also cleave one or more nucleic acid molecules containing homologous arms used in the methods described herein. In some embodiments, one or more endonucleases are selected from the group consisting of CRISPR/Cas endonucleases and one or more guide nucleic acids (gNA), one or more zinc finger nucleases (ZFN), or one or more transcription activator-like effector nucleases (TALEN). In some embodiments, one or more CRE recombinases are used to generate double-strand breaks in the template and target chromosomes to produce chromosomal rearrangements.

Different molecules are capable of introducing double-stranded and/or single-stranded breaks into genomic nucleic acids. The nucleases disclosed herein include, but are not limited to, homing endonucleases, restriction endonucleases, zinc finger nucleases or zinc finger nickases, meganickases or large-scale nucleases, transcription activator-like effector (TALE) nuclease-guided, particularly nucleic acid-guided, nucleases or nickases, such as RNA-guided nucleases, DNA-guided nucleases, megaTAL nucleases, BurrH nucleases, their modified or chimeric forms or variants, and combinations thereof. RNA-guided nucleases or RNA-guided nickases are optionally part of a CRISPR-based system.

Nucleases are capable of cleaving phosphodiester bonds between monomers of nucleic acids. Many nucleases participate in DNA repair by recognizing damage sites and cleaving them from the surrounding DNA. These enzymes can be part of a complex. Endonucleases are nucleases that act on the central region of the target molecule. Deoxyribonucleases act on DNA. Many nucleases involved in DNA repair are not sequence-specific. However, in this specification, sequence-specific nucleases are preferred. In some embodiments, one or more sequence-specific nucleases are specific to fairly large nucleotide strings (such as 10 or more nucleotides, or 15, 20, 25, 30, 35, 40, 45, or even 50 or more nucleotides) in the target genome, with the range of 5-50, 10-50, 15-50, 15-40, and 15-30 nucleotides as target sequences in the target genome being preferred. The larger this "recognition sequence" is, the fewer target sites there are in the genome, and the more specific the cleavage formed by the nuclease in the genome becomes, thus making the cleavage site-specific. Site-specific nucleases typically have fewer than 10, 5, 4, 3, 2, or just one (1) target site in the genome. Nucleases that have been engineered to alter one or more genomic nucleic acids (including by cutting specific genomic target sequences) are referred to herein as engineered nucleases. CRISPR-based systems are one type of engineered nuclease. However, such engineered nucleases can be based on any nuclease described herein.

Endonucleases that recognize sequences of more than 12 base pairs are called macronucleases. Macronucleases/-nicking enzymes are endonucleases characterized by large recognition sites (e.g., 12 to 40 base pairs, such as double-stranded DNA sequences of 20 to 40 or 30 to 40 base pairs); therefore, this site may only appear once in any given genome.

"Homing endonucleases" are a type of broad-spectrum nuclease, a double-stranded DNAase with large asymmetric recognition sites and coding sequences that typically embed introns or intepids. The recognition sites of homing endonucleases are extremely rare in the genome, resulting in cleavage at very few locations, sometimes even at a single location in the genome (WO2004067736, see also US Patent No. 8,697,395B2).

Zinc finger nucleases/-nickases (ZFNs) are artificial restriction endonucleases created by fusing a zinc finger DNA-binding domain with a DNA-cutting domain. The zinc finger domain can be engineered to target specific desired DNA sequences.

RNA-guided nucleases/-nickases, particularly endonucleases, include, for example, Cas9 or Cpf1. CRISPR systems have been described in detail. Any CRISPR-based system is part of this disclosure. When using one or more additional RNA-guided endonucleases, suitable guide RNA, sgRNA, or crRNA, or other suitable RNA sequences, can be used that interact with the RNA-guided endonuclease and target genomic target sites in the genomic nucleic acid.

As used herein, the term "CRISPR-associated protein" or "CRISPR/Cas" protein refers to a nucleic acid-guided DNA endonuclease associated with the CRISPR (clustered, regularly spaced short palindromic repeats) type II adaptive immune system found in certain bacteria, such as Streptococcus pyogenes and others. CRISPR/Cas proteins, such as Cas9, are not limited to wild-type (wt) proteins found in bacteria. CRISPR/Cas proteins containing mutations to wild-type CRISPR/Cas sequences or derivatives thereof are considered to be within the scope of this disclosure. The primitive type II CRISPR system from Streptococcus pyogenes comprises the Cas9 protein and a guide RNA consisting of two RNAs: mature CRISPR RNA (crRNA) and partially complementary trans-acting RNA (tracrRNA). Cas9 unwinds foreign DNA and checks for sites complementary to the 20-base-pair spacer region of the guide RNA. Cas9 targeting has been simplified, and most Cas-based systems have been engineered to require only one or two chimeric guide RNAs or a single guide RNA (chiRNA, often simply referred to as guide RNA, gRNA, or sgRNA), which is generated by the fusion of crRNA and tracrRNA. The spacer region can be engineered as needed.

As used herein, the term "Cas9 coding sequence" refers to a polynucleotide capable of being transcribed and/or translated (according to the genetic code that functions in the host cell/host mammal) to produce the Cas9 protein. The Cas9 coding sequence can be DNA (such as a plasmid) or RNA (such as mRNA).

As used herein, the term CRISPR/Cas ribonucleoprotein refers to a protein/nucleic acid complex composed of a CRISPR/Cas protein and an associated guide RNA. For example, Cas9 ribonucleoprotein refers to Cas9 complexed with its associated guide RNA.

In some embodiments, the nuclease is an RNA-guided nuclease. Non-limiting examples of RNA-guided nucleases (including nucleic acid-guided nucleases) used in this disclosure include, but are not limited to, CasI, CasIB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, CasX, CasY, Cas12a(Cpf1), Cas12b, Cas13a, CsyI, Csy2, Csy3, CseI, Cse2, CscI, Csc2, and Csa5. Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, CmrI, Cmr3, Cmr4, Cmr5, Cmr6, CsbI, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, CsfI, Csf2, Csf3, Csf4, Cms1, C2c1, C2c2, C2c3 or their homologs, direct homologs or modified forms.

“megaTAL nucleases/-cleaving enzymes” refers to engineered nucleases and engineered broad-based nucleases or engineered homing endonucleases that contain engineered TALE DNA-binding domains. TALE DNA-binding domains can be designed to bind DNA at virtually any locus of a nucleic acid sequence in the genome, and if such a DNA-binding domain is fused with an engineered broad-based nuclease, it cleaves the target sequence. Illustrative examples of megaTAL nucleases and the design of TALE DNA-binding domains are disclosed, for example, by Boissel et al. (MegaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering (2013), Nucleic Acids Research 42(4):2591-2601) and the references cited herein, all of which are incorporated herein by reference in their entirety. megaTAL nucleases optionally include one or more adapters and/or additional functional domains, such as C-terminal domain (CTD) peptides, N-terminal domain (NTD) peptides, terminal processing enzymatic domains displaying 5-3' exonuclease or 3-5' exonuclease, or other non-nuclease domains, such as helicase domains.

Transcription activator-like effector (TALE) nucleases/-cleavage enzymes are restriction endonucleases that can be engineered to cleave specific DNA sequences. TALEs can be engineered to bind to virtually any desired DNA sequence, so that when they bind to DNA cleavage domains, the DNA can be cleaved at a specific location.

The “TALE DNA-binding domain” is the DNA-binding portion of transcription activator-like effectors (TALE or TAL-effectors) that mimics plant transcription activators to manipulate the plant transcriptome. In some implementations, the TALE DNA-binding domains considered are de novo engineered or derived from naturally occurring TALEs, including but not limited to AvrBs3 from Xanthomonas campestris pv. vesicatoria, Xanthomonas gardeneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforans, Xanthomonas alfalfa, Xanthomonas citri, Xanthomonas euvesicatoria, and Xanthomonas oryzae, as well as brg11 and hpx17 from Ralstonia solanacearum. Illustrative examples of TALE proteins for deriving and designing DNA-binding domains are disclosed in U.S. Patent No. 9,017,967 and the references cited therein, all of which are incorporated herein by reference in their entirety.

"BurrH-nuclease" refers to a fusion protein with nuclease activity containing a modular base/base-specific nucleic acid binding domain (MBBBD). These domains are derived from proteins of the bacterial endocellular symbiont Burkholderia Rhizoxinica or other similar proteins identified from marine organisms. By combining different modules of these binding domains, the modular base/base binding domain can be engineered to have binding properties to specific nucleic acid sequences, such as DNA-binding domains. Therefore, such engineered MBBBDs can be fused with nuclease catalytic domains to cleave DNA at virtually any site in the nucleic acid sequence of the genome. Illustrative examples of BurrH-nuclease and MBBBD design are disclosed in WO 2014/018601 and US2015225465 A1, and the references cited therein, all of which are incorporated herein by reference in their entirety.

Relevant aspects of this disclosure provide nucleic acid molecules, such as vectors, suitable for generating CRISPR/Cas-mediated double-strand breaks (DSBs) in cells. In some embodiments, the vector comprises a sequence encoding a CRISPR/Cas protein, such as Cas9, and a sequence of a guide nucleic acid (Cas9 single guide RNA, or sgRNA) operatively linked to a promoter suitable for their expression in cells, as well as other vector components such as origin of replication and selection markers. In some embodiments, the cells are embryonic stem cells or embryonic hybrid stem cells as described herein.

According to this disclosure, homologous recombination is facilitated by double-strand breaks (DSBs) generated by a nuclease. In some embodiments, the nuclease comprises CRISPR/Cas9 and one or more single guide RNAs (referred to as "sgRNA" or "gRNA"). Those skilled in the art or of ordinary skill will be able to select the guide RNA having a target located flanking the template sequence and the target sequence, or a target sequence located at the target site, as described above for nuclease sites.

In some embodiments, the enzyme can be introduced by introducing a nucleic acid molecule (such as one or more vectors or coding sequences encoding CRISPR/Cas proteins) and one or more sgRNAs. In some embodiments, the vector or coding sequence encoding the CRISPR/Cas protein is CRISPR/Cas mRNA. In some embodiments, the vector or coding sequence encoding the CRISPR/Cas protein is a vector such as a plasmid containing a DNA sequence encoding the CRISPR/Cas protein and gRNA. In some embodiments, the CRISPR/Cas protein is Cas9.

In some embodiments, isolated CRISPR/Cas proteins can be directly introduced into cells (e.g., fertilized eggs or ES cells, via microinjection or electroporation). The CRISPR/Cas protein may be in the form of a CRISPR/Cas ribonucleoprotein, which is a CRISPR/Cas protein/gNA (guide nucleic acid) complex. Alternatively, the CRISPR/Cas protein may not contain any gNA, allowing the co-introduction of the CRISPR/Cas protein and one or more gNAs into fertilized eggs or ES cells to allow in situ formation of the CRISPR/Cas protein/gNA complex within the cell. In some embodiments, the CRISPR/Cas protein and gNA are encoded by a vector introduced into cells via transfection, electroporation, or transduction. In some embodiments, the CRISPR/Cas protein is Cas9.

In order to be used as a nuclease in the methods disclosed herein, the CRISPR/Cas protein needs to form a functional complex with gRNA.

According to some implementation schemes, multiple gNAs are used, each gNA targeting a specific CRISPR/Cas cleavage site. For example, four gNAs can be used: two with target sequences specific to the gNA target sequence on either side of the template sequence, and two with target sequences specific to the gNA target sequence on either side of the target sequence. Alternatively, three gNAs can be used: one with a target sequence specific to the gNA target sequence at the target site, and two with target sequences specific to the gNA target sequence on either side of the template sequence. As yet another example, two gNAs can be used: one with a target sequence specific to the gNA target sequence adjacent to the template sequence, and one with a target sequence specific to the gNA target sequence adjacent to the target sequence.

Preferably, regardless of the number of gNAs used to generate the DSB, in some embodiments each gNA is selected independently based on its proximity to the 5' and 3' ends of the template and target sequences or the target location.

gRNAs can be selected and designed based on user input (such as target genome and sequence type) using well-known principles or online tools. Generally, for Cas9, gRNAs are short synthetic RNAs consisting of a "scaffold" sequence necessary for Cas9 binding and a user-defined "spacer" or "target" sequence of approximately 20 nucleotides, which defines the genomic target to be bound or modified by the target sequence. For simplicity, "gRNA targeting a Cas9 cleavage site" means that the spacer or target sequence of the gRNA is designed to bind to and cleave the genomic target sequence at the cleavage site.

The length of the guide nucleic acid (including gRNA and gDNA) according to this disclosure can be any number of nucleotides more than 10 nucleotides, including 10-50 nucleotides, 10-40 nucleotides, 10-30 nucleotides, 10-20 nucleotides, 15-25 nucleotides, 16-24 nucleotides, 17-23 nucleotides, 18-22 nucleotides, 19-21 nucleotides and 20 nucleotides.

Preferably, the target sequence is unique enough that it theoretically binds to a unique genomic target sequence (compared to the rest of the genome). The target should be located immediately upstream (or 5') of the prespacer motif (or "PAM" sequence). The PAM sequence is absolutely necessary for target binding, and the exact sequence depends on the species of Cas9. In the most widely used Streptococcus pyogenes Cas9, the PAM sequence is 5′-NGG-3′ ("N" represents any of the four standard nucleotides). Other PAM sequences for other Cas9s in different species are known in the art. See the exemplary PAM sequences listed in Table 4 below.

Table 4. PAM sequences

Types/Variants of Cas9 PAM sequence Streptococcus pyogenes (SP); SpCas9 NGG SpCas9 D1135E variant NGG (Reduced NAG Binding) SpCas9 VRER variant NGCG SpCas9 EQR variant NGAG SpCas9 VQR variant NGAN or NGNG Staphylococcus aureus (SA); SaCas9 NNGRRT or NNGRR(N) Neisseria meningitidis (NM) NNNNGATT Streptococcus thermophilus (ST) NNAGAAW Treponema denticulata (TD) NAAAAC

The Cas9-gRNA complex will bind to any target genome sequence containing the PAM, but if there is sufficient homology between the gRNA spacer region and the target genome sequence, Cas9 will only cleave the target genome sequence. The final result of Cas9-mediated DNA cleavage is a double-strand break (DSB) at a cleavage site approximately 3-4 nucleotides upstream of the PAM sequence within the target genome sequence.

In some embodiments, double-strand breaks are generated on or between the target sequence. For example, in those embodiments where the target chromosome contains a target location (such as a location where the template sequence will be inserted with little or no deletion of the target chromosome), then the double-strand break is generated at the target location. Exemplary target locations include cleavage sites of any nucleases described herein. As another example, in those embodiments where the target chromosome contains a target sequence (such as a sequence that will be replaced or deleted due to the insertion of the template sequence), then the double-strand break is generated on either side of the target sequence (i.e., the 5' and 3' of the target sequence).

In some implementations, the cleavage site of any selected endonuclease (e.g., the gNA target sequence) is within approximately 10 bp, approximately 20 bp, approximately 30 bp, approximately 50 bp, approximately 70 bp, approximately 100 bp, approximately 200 bp, approximately 300 bp, approximately 400 bp, or approximately 500 bp of the target sequence or location.

In some implementations, the cleavage site of any selected endonuclease (e.g., the gNA target sequence) is within approximately 100 bp, approximately 200 bp, approximately 300 bp, approximately 400 bp, approximately 500 bp, approximately 600 bp, approximately 700 bp, approximately 800 bp, approximately 900 bp, approximately 1,000 bp, approximately 1,100 bp, approximately 1,200 bp, approximately 1,300 bp, approximately 1,400 bp, approximately 1,500 bp, approximately 1,600 bp, approximately 1,700 bp, approximately 1,800 bp, approximately 1,900 bp, or approximately 2,000 bp of the template sequence.

In some implementations, double-strand breaks are repaired via at least one DNA repair pathway selected from the group consisting of: excision, mismatch repair (MMR), nucleotide excision repair (NER), base excision repair (BER), canonical non-homologous end joining (canonical NHEJ), alternative non-homologous end joining (ALT-NHEJ), canonical homologous directed repair (canonical HDR), alternative homologous directed repair (ALT-HDR), microhomology-mediated end joining (MMEJ), blunt end joining, synthesis-dependent microhomology-mediated end joining, single-strand annealing (SSA), Holliday junction model or double-strand break repair (DSBR), synthesis-dependent strand annealing (SDSA), single-strand break repair (SSBR), trans-damage synthetic repair (TLS), and interstrand crosslinking repair (ICL), as well as DNA/RNA processing.

Recycling of engineered chromosomes

This disclosure provides methods for recovering the engineered chromosomes described herein and transferring the engineered chromosomes into a cellular environment suitable for downstream applications. In some embodiments, the recovery of the engineered chromosomes described herein includes microcell-mediated chromosome transfer (MMCT).

Microcell-mediated chromosome transfer (MMCT) is a technique for fusing microcells prepared from donor cells with recipient cells. Through this technique, specific (exogenous) DNA (e.g., chromosomes) from the donor cells can be transferred to the recipient cells. Microcells are typically prepared by treating donor cells with colchicine, although other methods can also be used, and such methods are also considered to be within the scope of this disclosure.

An exemplary MMCT protocol includes culturing cells containing engineered chromosomes in a cell culture medium containing at least one micronucleation inducer under conditions sufficient to induce micronucleation, thereby generating micronucleated cells, and collecting the micronucleated cells. Exemplary micronucleation inducers include, but are not limited to, microtubule polymerization inhibitors, microtubule depolymerization inhibitors, and spindle checkpoint inhibitors. Exemplary micronucleation inducers known in the art include, but are not limited to, colchicine, vincristine, or combinations thereof. For example, cells can be treated with 0.05 μg/mL to 0.25 μg/mL to induce micronucleation.

Micronucleated cells can be recovered using any suitable method known in the art, including centrifugation and filtration.

Therefore, this disclosure provides a method for recovering engineered chromosomes, the method comprising exposing cells to colchicine under conditions sufficient to induce micronucleation and collecting micronucleated cells using centrifugation.

In some implementations, the engineered chromosome contains one or more markers, such as selection markers or detectable markers introduced when the chromosome is engineered using a template sequence. These markers can be used to track the engineered chromosome and select cells containing the engineered chromosome after fusion with the aforementioned micronucleated cells.

Therefore, this disclosure provides a method for generating embryonic stem cells, comprising: (a) fusing micronuclear cells containing engineered chromosomes generated by the method of this disclosure with ES cells, wherein (i) the ES cells contain chromosomes homologous to the engineered chromosomes, the homologous chromosomes containing a first fluorescent protein operatively linked to a promoter capable of expressing a fluorescent protein in the ES cells, and (ii) at least one subpopulation of micronuclear cells containing engineered chromosomes, wherein the engineered chromosomes contain a second fluorescent protein different from the first fluorescent protein, the second fluorescent protein being operatively linked to a promoter capable of expressing a fluorescent protein in the ES cells; (b) selecting ES cells expressing both the first and second fluorescent proteins; (c) culturing the ES cells selected in step (c) until at least one subpopulation of ES cells loses the homologous chromosome; and (d) selecting ES cells expressing the second fluorescent protein but not the first fluorescent protein. In some embodiments, the ES cells are mouse, rat, rabbit, guinea pig, hamster, sheep, goat, donkey, cattle, horse, camel, chicken, or monkey ES cells. In some embodiments, the ES cells are mouse ES cells. In some embodiments, the ES cells are rat ES cells. In some implementations, the ES cells are monkey ES cells.

While the methods for generating embryonic stem cells described above use two different fluorescent proteins as markers, those skilled in the art will understand that other markers may be suitable, provided the markers on the engineered chromosomes and homologous chromosomes are different. For example, the two different selection markers described herein, as well as two different surface molecules that can be recognized by labeled antibodies or conjugated to selection markers such as gold particles, can be used, allowing selection by centrifugation. As another example, in addition to fluorescent proteins as markers, puromycin and hygromycin/thymidine kinase (TK) markers can also be used for positive-negative selection in this step. When thymidine kinase is expressed in the presence of specific thymidine analogs, these analogs are converted into toxic compounds that kill cells. For example, a puromycin resistance marker and a hygromycin/TK marker are knocked into the same location on two chromosomes, and double-positive monoclonal clones are selected by culturing in puromycin and hygromycin. After several days of culture, clones that have lost a copy of a chromosome carrying the hygromycin/TK marker are selected using puromycin and thymidine kinase.

In some embodiments, a method for generating embryonic stem cells includes (a) fusing micronucleated cells containing engineered chromosomes generated by the methods of this disclosure with ES cells, wherein (i) the ES cells contain chromosomes homologous to the engineered chromosomes, the homologous chromosomes containing a first marker, and (ii) at least one subpopulation of micronucleated cells contains engineered chromosomes, and wherein the engineered chromosomes contain a second marker different from the first marker; (b) selecting ES cells expressing both the first and second markers; (c) culturing the ES cells selected in step (c) until at least one subpopulation of ES cells loses the homologous chromosomes; and (d) selecting ES cells expressing the second marker but not the first marker.

Micronucleated cells can be fused with ES cells using any suitable method. Fusion methods include, in particular, electrofusion, virus-induced fusion, and chemically induced fusion, such as by adding PEG1000 to the cells.

Considering the inherent instability of trisomy resulting from the aforementioned method of recovering engineered chromosomes, culturing cells generated by fusion with micronucleated cells for at least 5, 7, 10, or 14 days is sufficient to obtain cells that have lost the homologous chromosome corresponding to the engineered chromosome. Alternatively, a selection scheme employing a negative selection marker, such as a marker located on the homologous chromosome, can be used, whose expression kills cells when exposed to the selection scheme. In some embodiments, cell selection in steps (b) and (d) includes fluorescence-activated cell sorting (FACS). For example, the cells may be FACS-sorted cells that express a second fluorescent protein for labeling the engineered chromosome but not a first fluorescent protein for labeling the homologous chromosome.

cell

This invention provides cells for use in the methods of this disclosure. In some embodiments, the cells include embryonic stem (ES) cells, hybrid embryonic stem (EHS) cells, or fertilized egg cells. This disclosure also provides cells comprising engineered chromosomes produced by the methods of this disclosure. This disclosure provides methods for isolating, fusing, and culturing the cells described herein.

Therefore, this disclosure provides a method for fusing cells to generate the EHS cells described herein. Cell fusion has become possible through chemical, biological, and physical means. Examples of these techniques include polyethylene glycol (PEG) fusion, fusagenic virus fusion, and electrofusion.

The ES cells used in the methods of this disclosure can be obtained from a variety of sources and can be primary isolated ES cells or artificially or naturally generated ES cell lines. The ES cells may also be genetically modified before or after cell fusion to generate the EHS cells of this disclosure, or before or after the methods described herein, to introduce useful traits, such as the expression of one or more markers.

A common technique is chemical fusion using, for example, PEG. This technique is particularly successful in generating hybridomas. The fusion probability can be increased by exposing cells to a strong electric field for a very short time. Before exposure to the electric field, chemical agents can be used in a suspension to achieve the linkage and proximity of the desired type of cell pair (i.e., two types of EH cells).

Electrofusion of cells involves bringing cells together tightly and exposing them to an alternating electric field. Under appropriate conditions, the cells are pushed together, their cell membranes fuse, and they form fused cells or hybrid cells. Electrofusion of cells and apparatus for performing electrofusion are described, for example, in U.S. Patent Nos. 4,441,972, 4,578,168, and 5,283,194, and International Patent Application No. PCT/AU92/00473. Typically, the method involves selecting cells and placing them in a fluid-filled chamber used as a cell fusion chamber. Single cell pairs may participate in the fusion process, i.e., single-cell fusion, or bulk fusion may occur in two populations, each containing two or more cells. Bulk fusion can be a mini-bulk fusion involving about 2 to about 1000 cells, or a macro-bulk fusion involving more than about 1000 cells. Fusion can be promoted by chemical means (such as in the presence of PEG), biological means (such as in the presence of a fusion virus), or electrical means (i.e., electrofusion). Fusion can also include a combination of these techniques. Treatment of cells with cytokines such as interleukin-3 (IL-3) can also promote fusion.

Following cell fusion, fused cells (fusate cells) or hybrid cells are obtained, containing the nuclei of at least two cells enclosed in a fusion lipid bilayer derived from the cells involved in the fusion. Nuclear fusion produces hybrid cells with an abnormal number of chromosomes, which may be tetraploid or contain fewer or more chromosomes. Hybrid cells possess the ability to divide and proliferate under appropriate culture conditions.

In some embodiments, EHS cells are generated by electrofusion. For example, human and mouse, human and rat, or human and monkey ES cells can be fused by electrofusion. In some embodiments, two EHS cells from two different species undergo electrofusion to generate EHS cells, said species being selected from the group consisting of: human, mouse, rat, rabbit, guinea pig, hamster, sheep, goat, donkey, cattle, horse, camel, chicken, and monkey.

Typically, once fusion occurs, the resulting hybrid cells are recovered in a suitable enriched medium and then amplified in culture for use in the methods disclosed herein. The recovery medium should contain factors that allow the cell fusion to recover after fusion stress. This supplement may contain a high percentage (e.g., 20%) of fetal bovine serum.

Hybrid cells generated through cell fusion can contain unique cell surface markers that can be used to select these cells and monitor fusion events.

In some embodiments, the cells of this disclosure contain one or more genetic modifications, such as the introduction of markers described herein. Genetic modifications can be performed by any suitable method known in the art. For example, cells can be modified by transfection, transduction, electroporation, lipid transfection, etc.

Transfection, as used herein, refers to the introduction of nucleic acids (including naked nucleic acids, purified nucleic acids, or vectors carrying specific nucleic acids) into cells, particularly eukaryotic cells, including mammalian cells. Any known transfection method may be used within the scope of this disclosure. Some of these methods involve enhancing the permeability of biological membranes to carry nucleic acids into cells. Prominent examples are electroporation, microporation, and lipid transfection. These methods may be used alone or supported by acoustic, electromagnetic, and thermal energy, chemipore enhancers, pressure, etc., to selectively increase the flux rate of nucleic acids into host cells. Other transfection methods are also within the scope of this disclosure, such as vector-based transfection, including lipid transfection or virus-based (also known as transduction) and chemical transfection. However, any method for carrying nucleic acids into cells may be used. Transiently transfected cells will carry/express the transfected RNA/DNA for a short period and will not pass it on. Stably transfected cells will continuously express and pass on the transfected DNA: the exogenous nucleic acid has been integrated into the cell's genome.

Many viruses have been used as gene transfer vectors or as the basis for preparing gene transfer vectors, including lactopolyviruses, adenoviruses, vaccinia viruses, adeno-associated viruses, lentiviruses, Sindbis and Semlikie forest viruses, as well as avian and human retroviruses.

Gene transfer can be achieved through chemical techniques (including calcium phosphate coprecipitation), mechanical techniques (e.g., microinjection), membrane fusion-mediated transfer via liposomes, direct DNA uptake, and receptor-mediated DNA transfer. Virus-mediated gene transfer can be combined with direct in vivo gene transfer using liposomes, allowing viral vectors to be directed to specific cells. Alternatively, retroviral vector-producing cell lines can be injected into specific tissues. Injection of producing cells provides a continuous source of vector particles.

This disclosure provides methods for culturing the cells of this disclosure. Many stem cell culture media or growth environments are envisioned in the embodiments described herein, including well-defined media, conditioned media, feeder-free media, serum-free media, etc. As used herein, the term "growth environment" is equivalent to an environment in which undifferentiated or differentiated stem cells (e.g., embryonic stem cells) will proliferate in vitro. The environment is characterized by the culture medium in which the cells are cultured and supporting structures (such as a matrix on a solid surface, if present). Methods for culturing or maintaining cells are also described in PCT/US2007/062755, U.S. Application No. 11/993,399, and U.S. Application No. 11/875,057.

Basic cell culture media are known in the art and are commercially available. Exemplary basic cell culture media include, but are not limited to, media based on DMEM, CMRL, or RPMI.

The cell culture methods disclosed herein may use cell culture media containing serum or without serum. The cell culture media may also contain one or more supplements or other culture medium components known in the art, such as B27 supplements, insulin, glucose, growth factors such as EGF and FGF, and cytokines.

The term "feeder cells" refers to a cell culture that grows in vitro and secretes at least one factor into a culture medium, which can be used to support the growth of another target cell in the culture. As used herein, the term "feeder cell layer" may be used interchangeably with the term "feeder cells." Feeder cells may comprise a monolayer, wherein the feeder cells cover the surface of the culture dish in a complete layer before growing on top of each other, or may comprise clusters of cells. In a preferred embodiment, the feeder cells comprise an adherent monolayer.

Similarly, embodiments in which ES or EHS cell cultures or aggregate suspension cultures are grown under defined conditions or culture systems without the use of feeder cells are "cupffer-free". A feeder-free method is also described in U.S. Patent No. 6,800,480. In some embodiments, ES or EHS cells can be cultured in a two-dimensional or three-dimensional environment. In U.S. Patent No. 6,800,480, the extracellular matrix is prepared by culturing fibroblasts, lysing them in situ, and then washing the remaining lysate. Alternatively, in U.S. Patent No. 6,800,480, the extracellular matrix can also be prepared from isolated matrix components or combinations of components selected from: collagen, placental matrix, fibronectin, laminin, merosin, tendinin, heparin sulfate, chondroitin sulfate, dermatan sulfate, agglutinin, biglycan, thrombin-sensitive protein, hydatidin, and core proteoglycan.

In some implementations, the culture method or system does not contain animal-derived products. In other implementations, the culture method is xeno-free.

This disclosure considers differentiating ES cells containing the engineered chromosomes described herein into various cell types for a range of downstream applications. Multiple strategies can be used to induce ES cells to differentiate into various cell types in vitro, typically involving supplementing the cell culture medium with exogenous biochemical compositions that guide the recapitulation of endogenous developmental cell signals and direct cell-specific differentiation. Strategies for differentiating ES cells are discussed in Vazin and Freed, Restor Neurol Neurosci (2010) 28(4):589-603 (the contents of which are incorporated herein by reference).

For example, ES or EHS cell populations can be further cultured in the presence of certain supplemental growth factors to obtain cell populations that have already developed or will develop into different cell lineages, or that can be selectively reversed to develop into different cell lineages. The term "supplemental growth factor," used in its broadest sense, refers to substances that effectively promote ES cell growth, maintain cell survival, stimulate cell differentiation, and/or stimulate the reversal of cell differentiation. Additionally, supplemental growth factors can be substances secreted by feeder cells into their culture medium. These substances include, but are not limited to, cytokines, chemokines, small molecules, neutralizing antibodies, and proteins. Growth factors may also include intercellular signaling peptides that control cell development and maintenance, as well as tissue form and function. In a preferred embodiment, the supplemented growth factor is selected from the group consisting of: steel cytokines (SCF), oncogene M (OSM), ciliary neurotrophic factor (CNTF), interleukin-6 (IL-6) in combination with soluble interleukin-6 receptor (IL-6R), fibroblast growth factor (FGF), bone morphogenetic protein (BMP), tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

The progression of stem cells to various pluripotent cells and/or differentiated cells can be monitored by determining the relative expression of a characteristic gene or gene marker of a specific cell type compared to the expression of a secondary gene or control gene (e.g., a housekeeping gene). In some processes, the expression of certain markers is determined by detecting the presence or absence of the marker. Alternatively, the expression of certain markers can be determined by measuring the level of the marker present in the cells of a cell culture or cell population. In such processes, the measurement of marker expression can be qualitative or quantitative. One method for quantifying the expression of a marker produced by a marker gene is by using quantitative PCR (Q-PCR). Methods for performing Q-PCR are well known in the art. Other methods known in the art can also be used to quantify marker gene expression. For example, the expression of a marker gene product can be detected using an antibody specific to the target marker gene product.

Genetically modified animals

This disclosure provides transgenic animals (e.g., transgenic mice) containing engineered chromosomes of this disclosure and methods for their preparation.

The choice of a suitable method for preparing transgenic animals from ES cells or fertilized egg cells containing the engineered chromosomes described herein will depend on the animal and is known to those skilled in the art.

In an exemplary method, an ES cell containing engineered chromosomes is integrated into an embryo at the blastocyst stage, which is then implanted into a pregnant or pseudopregnant female and delivered at full term. The result is a chimeric animal. If the ES cell produces germ cells, the animal's offspring will be fully transgenic and carry the engineered chromosomes.

In some implementation schemes, the genetically modified animal is a mouse, rat, rabbit, guinea pig, hamster, sheep, goat, donkey, cow, horse, camel, chicken, or monkey.

In some embodiments, the transgenic animal is a mouse. In some embodiments, producing a transgenic mouse includes injecting ES cells into a diploid blastocyst, transferring the nucleus of the ES cells to an enucleated mouse embryo, or a tetraploid embryo for complementation.

In some embodiments, the method further includes transferring ES cells or fertilized eggs into pseudopregnant females. In mice, pseudopregnant female mice are prepared by mating 6-8 week old female mice in natural estrus with vasectomized male mice. Fertilized eggs that have been processed and transferred to pseudopregnant females on the same day can be removed from the culture, placed in a pre-warmed suitable culture medium (such as M2 medium), and transferred via the oviduct to pseudopregnant females (e.g., 9-11 weeks old) 0.5 days after mating.

Once an engineered chromosome is inserted into a host mammal using the methods of this disclosure, the presence of the engineered chromosome can be verified in the resulting transgenic animal (e.g., a mouse) or its offspring. Such verification typically involves one or more genotyping operations on animals that may carry the engineered chromosome, polymerase chain reaction amplification of the linker sequence, direct sequencing of certain DNA fragments (e.g., a template sequence), and genetic mapping. Such techniques are well known in the art.

This disclosure provides transgenic mice comprising engineered chromosomes of the present disclosure. In some embodiments, the transgenic mouse comprises one or more humanized genes, such as any of the genes described in Tables 1 and 2. In some embodiments, the animal model comprises more than one humanized gene (e.g., 1, 2, 5, 10, 20, 50, 100 or more genes). In some embodiments, the transgenic mouse comprises all or part of a humanized immunoglobulin gene. In some embodiments, the transgenic mouse comprises all or part of a humanized TCR subunit gene.

In some embodiments of the transgenic mice disclosed herein, mouse chromosome 12 contains a sequence representing the human IGH variable region replacing the mouse Igh variable region. In some embodiments, the mouse Igh variable region contains _VH , _DH , and JH1-6 gene segments and intercalated non-coding sequences. In some embodiments, the human IGH variable region contains _VH , _DH , and _JH1-6 gene segments and intercalated non-coding sequences. In some embodiments, the engineered chromosome is mouse chromosome 6, which contains a sequence representing the human IGK variable region replacing the mouse Igk variable region. In some embodiments, the mouse Igk variable region sequence contains sequences encoding mouse _Vk and _Jk1-5 gene segments and intercalated non-coding sequences. In some embodiments, the template sequence contains a human IGK variable region sequence. In some embodiments, the human IGK variable region sequence contains sequences encoding human _Vk and _Jk1-5 gene segments and intercalated non-coding sequences.

application

Downstream applications of cells containing the engineered chromosomes described herein and of transgenic animals are considered to be within the scope of this disclosure.

Exemplary downstream applications include basic and applied research on animal models of human diseases and conditions using animal models (e.g., mice, rats, or monkeys) that have been humanized for one or more human genes. Exemplary but non-limiting genes are described in Tables 1 and 2, which can be humanized by replacing the model animal homolog with a human homolog. Animal models of human diseases associated with chromosomal abnormalities (translocations, inversions, etc.) can also be prepared using the methods described herein. Any animal model requiring large-scale chromosomal rearrangements of fragments larger than 300 kB, such as a humanized mouse model of Duchenne malnutrition (DMD), or an animal model requiring large-scale insertions or replacements of arrays of up to hundreds of genes, is considered to be within the scope of this disclosure.

In some embodiments (e.g., those in which the Igh variable region of the animal has been humanized), the transgenic animals of this disclosure can be used to produce humanized antibodies. For example, such animals can produce specific B cells with human antibodies or humanized antibodies. In some embodiments (e.g., those in which the Igk or Igl variable region of the animal has been humanized), the transgenic animals of this disclosure can be used to produce humanized antibodies.

In some embodiments (e.g., those in which a template sequence containing an antibody or an antigen fragment thereof has been inserted into a target chromosome), the transgenic animals of this disclosure can be used to produce antibodies or antigen-binding fragments. For example, the transgenic animals can be used to produce single-chain variable fragments (scFv), nanobodies, bispecific antibodies, and multispecific antibodies. Such antibodies can be used for research or therapeutic purposes.

Exemplary downstream applications include those in which the engineered chromosome is not integrated into the transgenic animal. Conversely, as an example, ES cells containing engineered chromosomes differentiate into another cell type that can be used for research or therapeutic purposes.

Reagent test kit

This disclosure provides kits containing the nucleic acid molecules described herein. In some embodiments, the nucleic acid molecules are vectors, such as plasmids.

In some embodiments of the kits disclosed herein, the kit includes cells for use with the methods described herein, such as cryopreserved EHS cells. In some embodiments, the kit includes instructions for use of nucleic acid molecules and, optionally, the cells.

Example

Example 1: Establishment of Embryonic Hybrid Stem (EHS) Cells

The overall goal of this study was to obtain humanized mice with variable domains targeting the Igh and Igk genes. Humans and mice exhibit a high degree of similarity in antibody gene arrangement and expression, and the genomic organization of heavy chains is also similar in both. Therefore, humanized forms of the variable domains of the mouse Igh or Igk genes could be obtained by replacing approximately 3 MB of mouse genome sequence containing all _VH , _DH , and _JH gene segments with approximately 1 MB of continuous human genome sequence containing equivalent human gene segments (Figure 1).

The first step in generating humanized mouse Igh genes is to produce mouse embryonic hybrid stem (EHS) cells by fusing mouse embryonic stem (ES) cells with human ES cells to generate cells with both mouse and human Igh genes.

Following standard procedures provided by the electrofusion instrument manufacturer, engineered mouse cells expressing a neomycin resistance gene under the control of the PGK promoter were fused with engineered human ES cells expressing an mCherry marker under the control of the CAG promoter. The hybrid EHS cells were cultured for 7 days in mouse ES cell medium containing G418, and surviving cells were sorted using fluorescence activated cell sorting (FACS) based on mCherry expression levels (Figure 2). Positive cells were serially cultured in mouse ES cell medium containing G418, and single-cell clones were isolated into individual wells for growth. Genomic DNA was then extracted from each single-cell clone for genotyping. Specifically, three pairs of primers (Figure 3A) for the V, D, and J regions of the human immunoglobulin heavy chain (IGH) were used for PCR to confirm the presence of the target sequence in the EHS clones (Figure 3B). Only clones containing all three desired regions were retained for further experiments.

Example 2: Engineered humanized chromosomes

2.1. Establishment of EHC via HDR-mediated chromosome rearrangement (HMCR)

To obtain mouse embryonic hybrid stem (EHS) cells with humanized variable domains of the Igh gene, approximately 3 MB of the variable domain of the mouse Igh gene on chromosome 12 was replaced with approximately 1 MB of the variable domain of the human IGH gene on human chromosome 4 via HDR-mediated chromosome rearrangement (HMCR; Figure 4A).

Two plasmids were designed to mediate the HMCR process and are shown in Figure 4A. The 5’ HMCR plasmid was designed to mediate the replacement of the 5’ end of the mouse Igh gene with its human counterpart, while the 3’ HMCR plasmid mediates the replacement of the 3’ end of the mouse Igh gene with its human counterpart. The 5’ HMCR plasmid contains a 5’ arm homologous to the 5’ end of the mouse Igh gene, a 3’ arm homologous to the 5’ end of the human IGH gene, and a cassette of CMV-EGFP-polyA-PGK-purinemycin-poly inserted between the two homologous arms. Similarly, the 3’ HMCR plasmid contains a 5’ arm homologous to the 3’ end of the human IGH variable locus, a 3’ arm homologous to the 3’ end of the mouse Igh variable locus, and a cassette of PGK-hygromycin-polyA inserted between the two homologous arms (see Figure 4A). The length of the homologous arms is between 600 bp and 1000 bp. Simultaneously, four plasmids containing Cas9 and sgRNAs targeting the 5' and 3' ends of the Igh variable domain in mice and humans were designed (see Figure 4A; sgRNA targeting sequences are provided in Table 7). These six plasmids were co-transfected as circular plasmids into the EHS cells obtained in Example 1 using standard methods, and the resulting cells were cultured for 7 days in mouse ES cell culture medium containing puromycin and hygromycin. Surviving GFP-positive single clones were selected for further culture.

Genotyping was performed to identify the desired single clone with successful HMCR. For genotyping, four pairs of PCR primers were designed, as shown in Figure 5A. For the first primer pair, the forward primer was designed upstream of the 5' homologous arm of the mouse Igh 5' HMCR plasmid, and the reverse primer was located within the CMV promoter region (Figure 5A). For the second primer pair, the forward primer was located within the puromycin gene of the 5' HMCR plasmid, and the reverse primer was located downstream of the 5' homologous arm of the human IGH, within the human IGH sequence (Figure 5A). For the third primer pair, the forward primer was located upstream of the homologous arm of the 3' homologous region of the human IGH variable region, and the reverse primer was located within the PGK promoter region of the 3' HMCR plasmid (Figure 5A). For the last primer pair, the forward primer was located within the hygromycin gene of the 3' HMCR plasmid, and the reverse primer was located downstream of the 3' homologous sequence of the 3' HMCR plasmid, within the mouse Igh variable domain (Figure 5A). Each clone was amplified by PCR using each primer pair, and only clones that showed positive PCR products for all four genotyping tests were retained for further experiments. Of the 196 clones isolated in this step, 6 were identified as positive for all four PCR amplicons (Figure 5B).

To promote expression of the human IGH gene in EHS cells with successful HMCR, the 3' selection marker was deleted from the genome of positive clones via homology-directed repair (HDR) (Figure 4A), although non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homology-mediated end joining (HMEJ) methods can also be used. The above methods successfully created an engineered humanized chromosome (EHC) in EHS cells, in which the variable domain of the mouse Igh gene on mouse chromosome 12, containing the _VH , _DH , and _JH1-6 gene segments, was replaced with an equivalent human region by HMCR.

Tables 5 and 6 below provide plasmid sequences used to mediate the HMCR process.

Table 5. Exemplary 5' plasmid sequences for HMCR-mediated substitution of mouse Igh variable regions using corresponding human regions.

Table 6. Exemplary 3' plasmid sequences for HMCR-mediated substitution of mouse Igh variable regions using corresponding human regions.

Table 7. sgRNA Sequences

Table 7 provides sgRNA sequences having a PAM (NGG) sequence located at the 3' of the non-target strand of the sgRNA targeting sequence. Corresponding sgRNA targeting sequences without PAM are provided as SEQ ID NOS:14-17.

2.2. Establishment of EHC via CRE-Loxp-mediated Chromosomal Rearrangement (CMCR)

To obtain humanized mouse EHS cells with variable domains targeting their Igh genes, approximately 3 MB of variable domains of the mouse Igh gene on chromosome 12 were replaced with approximately 1 Mb of variable domains of the Igh gene on human chromosome 14 via CRE-Loxp-mediated chromosome rearrangement (CMCR; Figure 4B). Four plasmids were designed to mediate the CMCR process. The mouse Igh 5’ (pCMV-GFP-BGH PolyA-Loxp) and 3’ (BGH polyA-Loxp-511-hygromycin-BGH polyA-PGK-BSD-BGHPolyA) plasmids were designed to be inserted into the 5’ and 3’ ends of the mouse Igh variable locus, respectively. Simultaneously, human IGH 5’ (BGHpolyA-Loxp-Puro-BGH PolyA-PGK-neomycin-BGH PolyA) and 3’ (pCMV-BGP-BGH PolyA-PGK-Loxp-511) plasmids were designed to be inserted into the 5’ and 3’ ends of the human IGH variable locus, respectively (Figure 5). Transfected EHS cells were cultured for 7 days in mouse ES cell culture medium containing BSD and neomycin. Surviving GFP- and BFP- double-positive cells were selected for further culture. Genotyping was performed to identify the desired single clones that successfully integrated the aforementioned plasmids. Cre was transfected into successfully integrated EHS cells for CMCR, and successfully rearranged cells survived in culture medium containing puromycin and hygromycin. The surviving cells were then placed in bags for genotyping. To promote expression of the human IGH gene in EHS cells with successful CMCR, the 3’ selection marker was then deleted from the genome (Figure 5). Following the above process, engineered humanized chromosomes (EHCs) were successfully established by performing CMCR in EHS cells; the Igh gene on mouse chromosome 12 was humanized for their variable domains.

Example 3: Chromosomal replacement in mouse embryonic stem cells via microcell-mediated chromosome transfer

EHS cells with engineered humanized chromosomes (EHC) were obtained as described in Examples 1 and 2, and then the EHC was transferred to mouse ES cells via microcell-mediated chromosome transfer (MMCT) to establish mouse ES cells with humanized Igh gene variable domains.

EHS cells carrying EHC were treated with 0.2 μg/ml colchicine at 37°C for 48 hours. Prolonged mitotic arrest induced microcell formation, which was collected by centrifugation (Fig. 6). Simultaneously, mouse ES cells expressing the mCherry fluorescent label on chromosome 12 were obtained (Fig. 6). These cells were obtained by inserting a CMV-mCherry-polyA cassette into a copy of mouse chromosome 12.

Next, microcells were hybridized to mouse ES cells via electrofusion, and the resulting cells were sorted by FACS using GFP+ and mCherry+ labels to obtain GFP+ and mCherry+ mouse ES cells. GFP+ indicates that EHC was successfully transferred to mouse ES cells, while mCherry+ labeling indicates that the cells also carry mCherry+ chromosome 12. Positive cells were cultured in mouse ES cell culture medium for 2 weeks, and mCherry- and GFP+ mouse ES cells (i.e., cells that have lost the additional chromosome 12 labeled mCherry+) were sorted by FACS and cultured for 7 days. Individual clones were isolated into separate wells for growth and karyotype analysis, retaining clones with the correct karyotype. The result is humanized mouse ES cells targeting the variable region of their Igh gene.

Example 4: Generation of Igh humanized mice

According to standard procedures, humanized mouse ES cells with the variable region of the Igh gene obtained in Example 3 were injected into the blastocysts of the B6D2F1 (C57BL/6X DBA2) mouse strain. Alternatively, nuclear transfer or tetraploid embryo complementation can also be used to generate humanized mice.

Two and a half days post-mating (dpc), the injected blastocysts were transferred to the uterus of pseudopregnant ICR females. Igh humanized mice were identified by GFP expression levels under a fluorescence stereomicroscope, and GFP+ mice were further analyzed.

Next, a series of PCR experiments were designed to validate the Igh humanized mice. The first set of PCR experiments was designed to verify the integrity of the human IGH variable region. Five primer pairs targeting different regions of the human IGH variable region were designed (see Figure 7A, arrows indicate PCR primers 1-10). The Igh humanized mice showed positive PCR products for all five PCR primer pairs (Figure 7B). We also designed primers upstream and downstream of the human IGH variable region (Figure 7A), but no products were observed for any of our Igh humanized mice in any of the PCR experiments, while HEK293T showed the correct band of the PCR product (Figure 7B).

Fibroblasts were isolated from the tail of Igh humanized mice and used for fluorescence in situ hybridization (FISH). The FISH results showed that chromosome 12 of the Igh humanized mice contained a segment of human chromosome 14 (Figure 8A), indicating that the variable domain of the human IGH gene was successfully inserted in situ into mouse chromosome 12.

G-banding karyotype analysis was also performed to rule out any abnormal chromosomes (Figure 8B).

Genomic DNA was also extracted from Igh humanized mice and subjected to whole-genome sequencing (WGS) analysis. The WGS sequences were mapped onto a reference genome containing all mouse chromosomes and human chromosome 14. All variable domains of the human IGH gene ( _VH , _DH , and _JH gene segments) were covered by the whole-genome sequence reads. Furthermore, no off-target editing was found in other genomic regions (Figures 9A-9B).

Example 5: Production of IgK humanized mice

Humanized mice targeting the variable domain of the Igk gene were obtained using MASIRT (Figure 10). Using a similar method to that described above for the Igh gene, we also obtained Igk humanized mice. To validate the Igk humanized mice, we first performed PCR experiments to verify the integrity of the human IGK variable region. Five primer pairs were designed at different loci in the human IGK variable region (Figure 11A), and the obtained Igk humanized mice showed positive PCR products in all five experiments (Figure 11B). Primers upstream and downstream of the human IGK variable region were also designed (Figure 11A), but no products were observed in any of the PCR experiments for the obtained Igk humanized mice, while HEK293T showed the correct PCR band (Figure 11B). Finally, genomic DNA was extracted from the Igk humanized mice and whole-genome sequencing (WGS) analysis was performed.

Table 8. Exemplary 5' plasmid sequences for HMCR-mediated substitution of mouse Igk variable regions using corresponding human regions.

Table 9. Exemplary 3' plasmid sequences for HMCR-mediated substitution of mouse Igk variable regions using corresponding human regions.

Table 10. sgRNA sequences used to replace the mouse Igk variable region with the corresponding human region

sgRNA sequence SEQ ID NO Mice with PAM igk 5’ agtctctgctgcctacagcaNGG twenty four Mice with PAM igk 3’ agtccttgacagacagctcaNGG 25 People with PAM IGK 5' gcctatgatattacccagccNGG 26 People with PAM IGK 3' acccatgacctggccactgaNGG 27

Table 10 provides sgRNA sequences having a PAM (NGG) sequence located on the 3' of the non-target strand of the sgRNA targeting sequence. Corresponding sgRNA targeting sequences without PAM are provided as SEQ ID NOS:28-31.

Whole genome sequences containing all mouse chromosomes and a reference genome including human chromosome 2 were plotted. This shows that all variable domains of the human IGK gene ( _VH and _JH gene segments) are covered by the whole genome sequence. Furthermore, no off-target editing was found in other genomic regions (Figure 12).

Claims (105)

1. A method for producing engineered chromosomes, comprising: a. Provide cells containing a target chromosome with the target sequence and a template chromosome with the template sequence; b. Contact the cells with the following: i. A first nucleic acid molecule comprising, from 5' to 3', a 5' homologous arm, at least one first marker, and a 3' homologous arm, wherein the 5' homologous arm contains a nucleotide sequence upstream of the 5' end of the target sequence, and the 3' homologous arm contains a nucleotide sequence upstream of the 5' end of the template sequence; and ii. A second nucleic acid molecule comprising, from 5' to 3', a 5' homologous arm, at least one second marker, and a 3' homologous arm, wherein the 5' homologous arm contains a nucleotide sequence downstream of the 3' end of the template sequence, and the 3' homologous arm contains a nucleotide sequence downstream of the 3' end of the target sequence; c. Double-strand breaks are generated at or on either side of the target sequence, and at the 5' and 3' ends of the template sequence, thereby inserting the template sequence and the first and second markers into the target chromosome; and d. Select one or more cells that express the first and second markers. 2. The method of claim 1, wherein after inserting the template sequence, the first marker is located at the 5' end of the template sequence, and the second marker is located at the 3' end of the template sequence. 3. The method of claim 1 or 2, wherein the lengths of the 5' and 3' homologous arms of the first and second nucleic acid molecules are between about 20 bp and 2,000 bp, between about 50 bp and 1,500 bp, between about 100 bp and 1,400 bp, between about 150 bp and 1,300 bp, between about 200 bp and 1,200 bp, between about 300 bp and 1,100 bp, between about 400 bp and 1,000 bp, or between about 500 bp and 900 bp, or between about 600 bp and 800 bp. 4. The method of claim 1 or 2, wherein the lengths of the 5' and 3' homologous arms of the first and second nucleic acid molecules are between about 400 bp and 1,500 bp, between about 500 and 1,300 bp, or between about 600 and 1,000 bp. 5. The method of claim 1 or 2, wherein the lengths of the 5' and 3' homologous arms of the first and second nucleic acid molecules are between about 600 bp and 1,000 bp. 6. The method of any one of claims 1-5, wherein the template sequence has a length of at least 25 kilobase pairs (KB), at least 50 KB, at least about 100 KB, at least about 200 KB, at least about 400 KB, or at least about 500 KB. KB, at least 600KB, at least 700KB, at least 800KB, at least 900KB, at least 1 megabase pair (MB), at least 2MB, at least 3MB, at least 4MB, at least 5MB, at least 6MB, at least 7MB, at least 8MB, at least 9MB, at least 10MB, at least 15MB, at least 20MB, at least 25MB, at least 30MB, at least 40MB, at least 50MB, at least 60MB, at least 70MB, at least 80MB, at least 90MB, at least 100MB, at least 120MB, at least 140MB, at least 160MB, at least 180MB, at least 200MB, at least 220MB, or at least 250MB. 7. The method of any one of claims 1-5, wherein the length of the template sequence is between 50KB and 250MB. Between, between 50KB and 100MB, between 50KB and 50MB, between 50KB and 20MB Between, between 50KB and 10MB, between 50KB and 5MB, between 50KB and 3MB, between 50KB and 2MB, between 50KB and 1MB, between 100KB and 200MB, between 100KB and 100MB, between 100KB and 50MB, between 100KB and 20MB, between 100KB and 10MB, between 100KB and 5MB, between 100KB and 3MB, between 100KB and 2MB, between 100KB and 1MB, between 100KB and 50KB, between 100KB and 50MB, between 200KB and 200MB Between MB, between 200KB and 10MB, between 200KB and 5MB, between 200KB and 3MB Between MB, between 200KB and 2MB, between 200KB and 1MB, between 200KB and 500KB, between 500KB and 100MB, between 500KB and 50MB, between 500KB and 20MB, between 500KB and 10MB, between 500KB and 5MB, between 500KB and 3MB, between 500KB and 2MB, between 500KB and 1MB, between 1MB and 100MB Between 1MB and 50MB, between 1MB and 20MB, between 1MB and 10MB, between 1MB and 5MB, between 1MB and 3MB, between 1MB and 2MB, between 3MB and 100MB, between 3MB and 50MB, between 3MB and 20MB, between 3MB and 10MB, between 3MB and 5MB, between 5MB and 100MB, between 5MB and 5MB Between 50MB and 50MB, between 5MB and 20MB, between 5MB and 10MB, between 10MB and 10MB Between 100MB and 100MB, between 10MB and 50MB, or between 10MB and 20MB. 8. The method of any one of claims 1-5, wherein the length of the template sequence is between 200KB and 50MB. Between, between 1MB and 20MB, between 1MB and 10MB, between 1MB and 5MB, between 1MB and 3MB, between 3MB and 20MB, between 3MB and 10MB, between 3MB and 7MB, or between 3MB and 5MB. 9. The method of any one of claims 1-8, wherein generating the double-strand break in (c) comprises using CRISPR/Cas endonuclease and one or more guide nucleic acids (gNA), one or more zinc finger nucleases, one or more transcription activator-like effector nucleases (TALEN), or one or more CRE recombinases are used to induce the double-strand break. 10. The method of claim 9, wherein the CRISPR/Cas endonuclease comprises CasI, CasIB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, CasX, CasY, Cas12a(Cpf1), Cas12b, Cas13a, CsyI, Csy2, Csy3, CseI, Cse2, CscI, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, CmrI, Cmr3, Cmr4, Cmr5, Cmr6, CsbI, Csb2, Csb3, Csx17, CsxI4, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, CsfI, Csf2, Csf3, Csf4, Cms1, C2c1, C2c2 or C2c3 or their homologs, direct homologs or modified forms. 11. The method of claim 9, wherein the CRISPR/Cas endonuclease comprises Cas9, Cpf1, CasX, CasY, C2c1, C2c3 or their homologs, orthologs or modified forms. 12. The method of claim 9, wherein the CRISPR/Cas endonuclease comprises Cas9. 13. The method of any one of claims 10-12, wherein the gNA comprises a single guide RNA (sgRNA). 14. The method of any one of claims 1-13, wherein the target chromosome comprises, from 5' to 3', a 5' homologous arm sequence of a first nucleic acid molecule, a target sequence, and a 3' homologous arm sequence of a second nucleic acid molecule. 15. The method of any one of claims 1-14, wherein the template chromosome comprises, from 5' to 3', a 3' homologous arm sequence of a first nucleic acid molecule, a template sequence, and a 5' homologous arm sequence of a second nucleic acid molecule. 16. The method of any one of claims 1-15, wherein the target sequence comprises at least 1 gene, at least 2 genes, at least 3 genes, at least 5 genes, at least 10 genes, at least 20 genes, at least 30 genes, at least 40 genes, at least 50 genes, at least 100 genes, or at least 200 genes. 17. The method of any one of claims 1-16, wherein the target sequence comprises one or more genes homologous to one or more genes of the template sequence. 18. The method of any one of claims 1-17, wherein the template sequence comprises a naturally occurring sequence. 19. The method of claim 18, wherein the template sequence comprises one or more modifications to the naturally occurring sequence. 20. The method of claim 18, wherein the template sequence comprises at least 1 gene, at least 2 genes, at least 3 genes, at least 5 genes, at least 10 genes, at least 20 genes, at least 30 genes, at least 40 genes, at least 50 genes, at least 100 genes, or at least 200 genes. 21. The method of any one of claims 1-17, wherein the template sequence comprises an artificial sequence. 22. The method of claim 21, wherein the artificial sequence comprises a sequence encoding one or more antibodies or an antigen-binding fragment thereof. 23. The method of claim 22, wherein the one or more antibodies or antigen-binding fragments thereof comprise scFv, bispecific antibodies, or multispecific antibodies. 24. The method of any one of claims 1-23, wherein the target sequence is deleted by inserting the template sequence. 25. The method of claim 24, wherein: a. The target chromosome comprises, from 5' to 3', the 5' homologous arm sequence of the first nucleic acid molecule, the first sgRNA target sequence, the target sequence, the second sgRNA target sequence, and the 3' homologous arm sequence of the second nucleic acid molecule; and b. The template chromosome contains, from 5' to 3', a third sgRNA target sequence, a 3' homologous arm sequence of the first nucleic acid molecule, the template sequence, a 5' homologous arm sequence of the second nucleic acid molecule, and a fourth sgRNA target sequence. 26. The method of claim 25, wherein generating the double-strand break comprises contacting the cell with a CRISPR/Cas endonuclease and the first, second, third, and fourth sgRNAs. 27. The method of claim 26, wherein the first, second, third, and fourth sgRNAs comprise targeting sequences specific to the target sequences of the first, second, third, and fourth sgRNAs. 28. The method of claim 26, wherein contacting the cells with a CRISPR/Cas endonuclease and sgRNA comprises transfecting the cells with one or more nucleic acid molecules encoding the CRISPR/Cas endonuclease and the sgRNA. 29. The method of any one of claims 1-23, wherein inserting the template sequence comprises a sequence in which the target sequence is hardly deleted or is not deleted. 30. The method of claim 29, wherein inserting the template sequence disrupts one or more functions of the target sequence. 31. The method of claim 29 or 30, wherein inserting the template sequence disrupts the gene in the target sequence. 32. The method according to any one of claims 29-31, wherein a. The target chromosome comprises, from 5' to 3', the 5' homologous arm sequence of the first nucleic acid molecule, the first sgRNA target sequence, and the 3' homologous arm sequence of the second nucleic acid molecule; and b. The template chromosome contains, from 5' to 3', a second sgRNA target sequence, a 3' homologous arm sequence of the first nucleic acid molecule, a template sequence, a 5' homologous arm sequence of the second nucleic acid molecule, and a third sgRNA target sequence. 33. The method of claim 32, wherein generating the double-strand break comprises contacting the cell with a CRISPR/Cas endonuclease and first, second, and third sgRNAs. 34. The method of claim 33, wherein the first, second, and third sgRNAs comprise target sequences specific to the target sequences of the first, second, and third sgRNAs. 35. The method of claim 34 or 35, wherein contacting the cell with the CRISPR/Cas endonuclease and the sgRNA comprises transfecting the cell with one or more nucleic acid molecules encoding the CRISPR/Cas endonuclease and the sgRNA. 36. The method of any one of claims 1-35, wherein the first or second label comprises a fluorescent protein operatively linked to a promoter capable of expressing the fluorescent protein in the cell. 37. The method of claim 36, wherein the fluorescent protein comprises green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), dsRed, mCherry or tdTomato. 38. The method of claim 36, wherein the fluorescent protein comprises GFP. 39. The method of any one of claims 1-38, wherein the first mark further includes a selection mark. 40. The method of any one of claims 1-39, wherein the second mark further includes a selection mark. 41. The method of claim 39 or 40, wherein the selection marker is selected from the group consisting of: dihydrofolate reductase (DHFR), glutamine synthase (GS), puromycin acetyltransferase, blastomycin deaminase, histidine dehydrogenase, hygromycin phosphotransferase (hph), bleomycin resistance gene and aminoglycoside phosphotransferase (neomycin resistance). 42. The method of any one of claims 39-41, wherein the first and second marks are not the same selection marks. 43. The method of any one of claims 1-42, wherein the first label comprises GFP and puromycin acetyltransferase, the GFP being operatively linked to a promoter capable of expressing GFP in the cell, and the second label comprises hygromycin phosphotransferase. 44. The method of any one of claims 1 to 43, further comprising (e) deleting all or part of the first or second mark after step (d). 45. The method of claim 44, wherein deleting the first or second marker comprises inducing deletion with a CRISPR/Cas endonuclease and gNA, the gNA comprising a target sequence specific to the sequence encoding the marker. 46. The method of any one of claims 1-45, wherein the cell comprises a hybrid cell, an embryonic hybrid stem (EHS) cell, or a fertilized egg. 47. The method of claim 46, wherein the EHS cells are generated by fusing ES cells from any two species selected from the group consisting of: mice, rats, rabbits, guinea pigs, hamsters, sheep, goats, donkeys, cattle, horses, camels, chickens, and monkeys. 48. The method of claim 46, wherein the EHS cells are generated by fusing human embryonic stem cells with embryonic stem cells from a non-human species. 49. The method of claim 48, wherein the non-human species is a mouse, rat, rabbit, guinea pig, hamster, sheep, goat, donkey, cow, horse, camel, chicken, or monkey. 50. The method of claim 46, wherein the EHS cells are generated by fusing ES cells from any two different species, the species being selected from the group consisting of: mice, rats, rabbits, guinea pigs, hamsters, sheep, goats, donkeys, cattle, horses, camels, chickens, and monkeys. 51. The method of claim 46, wherein generating the hybrid cells comprises: a. Production of micronucleated human cells; and b. The micronucleated human cells are fused with cells from a non-human species to produce hybrid cells. 52. The method of claim 51, wherein the micronucleated human cells are generated by exposing human cells to colchicine under conditions sufficient to induce micronucleation and collecting the micronucleated cells by centrifugation. 53. The method of claim 51 or 52, wherein the non-human species is a mouse, rat, rabbit, guinea pig, hamster, sheep, goat, donkey, cow, horse, camel, chicken, or monkey. 54. The method of any one of claims 51-53, wherein the cells from the non-human species are ES cells and the hybrid cells are EHS cells. 55. The method of any one of claims 47-50, wherein the fusion comprises electrofusion, virus-induced fusion, or chemically-induced fusion. 56. The method of any one of claims 1-55, wherein the target sequence comprises a gene encoding an immunoglobulin or a T-cell receptor subunit. 57. The method of any one of claims 1-56, wherein the target chromosome comprises mouse chromosome 12 and the template chromosome comprises human chromosome 14, or wherein the target chromosome comprises mouse chromosome 6 and the template chromosome comprises human chromosome 2. 58. The method of claim 57, wherein the target sequence comprises a mouse Igh variable region sequence, a mouse Igk variable region sequence, and/or a mouse Igl variable region sequence. 59. The method of claim 58, wherein the mouse Igh variable region sequence comprises sequences encoding mouse _VH , _DH and _JH1-6 gene segments and interpolated non-coding sequences. 60. The method of any one of claims 57-59, wherein the template sequence comprises a human IGH variable region sequence, a human IGK variable region sequence, and/or a human IGL variable region sequence. 61. The method of claim 60, wherein the human IGH variable region sequence comprises sequences encoding human _VH , _DH and _JH 1-6 gene segments and interpolated non-coding sequences. 62. The method of any one of claims 1-61, further comprising recovering the engineered chromosome from the cell selected in step (d). 63. The method of claim 62, wherein recovering the engineered chromosome comprises exposing the cells to colchicine under conditions sufficient to induce micronucleation and collecting the micronucleated cells using centrifugation. 64. The method of any one of claims 1-63, wherein the first and second nucleic acid molecules are plasmids. 65. An engineered chromosome produced by any one of claims 1-64. 66. The engineered chromosome of claim 65, wherein the engineered chromosome is mouse chromosome 12 comprising a human IGH variable region sequence replacing the mouse Igh variable region, or wherein the engineered chromosome is mouse chromosome 6 comprising a human IGK variable region sequence replacing the mouse Igk variable region. 67. The engineered chromosome of claim 66, wherein the mouse Igh variable region comprises _VH , _DH and _JH 1-6 gene segments and intercalated non-coding sequences. 68. The engineered chromosome of claim 66 or 67, wherein the human IGH variable region comprises _VH , _DH and _JH 1-6 gene segments and intercalated non-coding sequences. 69. A cell comprising the engineered chromosome of any one of claims 64-68. 70. The cell of claim 69, wherein the cell is capable of hybridizing with mouse ES cells. 71. The cell of claim 69, wherein the cell is an embryonic stem (ES) cell, an embryonic hybrid stem (EHS) cell, or a fertilized egg. 72. The method of claim 68, wherein the cell is a micronucleated cell. 73. The cell of claim 72, wherein the EHS cell is a hybrid of human and mouse ES cells. 74. The cell of claim 72, wherein the ES cell is a mouse ES cell. 75. A method for generating mouse embryonic stem cells, the method comprising: a. Fusing micronucleated cells containing the engineered chromosome produced by any one of claims 1-64 with mouse ES cells, wherein: i. The mouse ES cells contain a chromosome homologous to the engineered chromosome, the homologous chromosome containing a first fluorescent protein operatively linked to a promoter capable of expressing the fluorescent protein in the ES cells, and ii. At least one subpopulation of the micronucleated cells comprises engineered chromosomes, and wherein the engineered chromosomes comprise a second fluorescent protein different from the first fluorescent protein, the second fluorescent protein being operatively linked to a promoter capable of expressing the fluorescent protein in the ES cells; b. Select ES cells expressing the first and second fluorescent proteins; c. Culture the ES cells selected in step (c) until at least one subgroup of the ES cells loses the homologous chromosome; and d. Select ES cells that express the second fluorescent protein but not the first fluorescent protein. 76. The method of claim 75, wherein culturing the cells in step (c) comprises culturing the cells for at least 5 days. One day, at least 7 days, at least 10 days, or at least 14 days. 77. The method of claim 75 or 76, wherein selecting the cells in steps (b) and (d) includes fluorescence activated cell sorting (FACS). 78. A mouse ES cell, which is generated by the method of any one of claims 75-77. 79. A transgenic mouse produced from mouse ES cells generated by the method of any one of claims 75-78. 80. The transgenic mouse of claim 79, wherein generating the transgenic mouse comprises injecting the ES cell into a diploid blastocyst, transferring from the nucleus of the ES cell to an enucleated mouse embryo, or complementing a tetraploid embryo. 81. The transgenic mouse of claim 79 or 80, wherein mouse chromosome 12 contains a human IGH variable region sequence replacing the mouse Igh variable region, or wherein mouse chromosome 6 contains a human IGK variable region sequence replacing the mouse Igk variable region. 82. The transgenic mouse of claim 81, wherein the mouse Igh variable region comprises _VH , _DH and _JH 1-6 gene segments and intercalated non-coding sequences. 83. The transgenic mouse of claim 81 or 82, wherein the human IGH variable region comprises _VH , _DH and _JH 1-6 gene segments and intercalated non-coding sequences. 84. A method for producing antibodies, comprising: a. The transgenic mouse of any one of claims 80-83 is attacked with an antigen, thereby producing a plurality of antibodies comprising human V, D, and J segments from the human IGH variable region; and b. Isolate antibodies specific to the antigen. 85. An antibody derived from an antibody produced by the method of claim 84. 86. The antibody of claim 85, wherein the antibody comprises a single-chain variable fragment (scFv), a bispecific antibody, or a multispecific antibody. 87. A method for generating chromosomal rearrangements, the method comprising: a. Provide cells containing a target chromosome with the target location and a template chromosome with the template sequence; b. Contact the cell with a nucleic acid molecule, the nucleic acid molecule comprising, from 5' to 3', a 5' homologous arm containing a nucleotide sequence upstream of the 5' end of the target site, a marker, and a 3' homologous arm containing a nucleotide sequence upstream of the 5' end of the template sequence; c. A double-strand break is created at the target site and the 5' end of the template sequence, thereby inserting the marker into the target chromosome at the 3' of the 5' homologous arm sequence, followed by insertion into the template sequence, thereby producing a chromosomal rearrangement; and d. Select one or more cells that express the marker. 88. The method of claim 87, wherein the lengths of the 5' and 3' homologous arms of the nucleic acid molecule are between about 20 bp and 2,000 bp, between about 50 bp and 1,500 bp, between about 100 bp and 1,400 bp, between about 150 bp and 1,300 bp, between about 200 bp and 1,200 bp, and between about 300 bp and 1,100 bp. Between approximately 400bp and 1,000bp, or between approximately 500bp and 900bp, or between approximately 600bp and 800bp. 89. The method of claim 87, wherein the lengths of the 5' and 3' homologous arms of the nucleic acid molecule are between about 400 bp and 1,500 bp, between about 500 bp and 1,300 bp, or between about 600 bp and 1,000 bp. 90. The method of claim 87, wherein the lengths of the 5' and 3' homologous arms of the nucleic acid molecule are between about 600 bp and 1,000 bp. 91. The method of any one of claims 87-90, wherein generating the double-strand break in (c) comprises using CRISPR/Cas endonuclease and at least one sgRNA, one or more zinc finger nucleases, one or more transcription activator-like effector nucleases (TALENs), or one or more CRE recombinases are used to induce the double-strand break. 92. The method of claim 91, wherein the CRISPR/Cas endonuclease comprises CasI, CasIB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, CasX, CasY, Cas12a(Cpf1), Cas12b, Cas13a, CsyI, Csy 2. Csy3, CseI, Cse2, CscI, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, CmrI, Cmr3, Cmr4, Cmr5, Cmr6, CsbI, Csb2, Csb3, Csx17, CsxI4, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, CsfI, Csf2, Csf3, Csf4, Cms1, C2c1, C2c2 or C2c3 or their homologs, direct homologs or modified forms. 93. The method of claim 91, wherein the CRISPR/Cas endonuclease comprises Cas9, Cpf1, CasX, CasY, C2c1, C2c3 or their homologs, direct homologs or modified forms. 94. The method of claim 91, wherein the CRISPR/Cas endonuclease comprises Cas9. 95. The method of any one of claims 91-93, wherein generating the double-strand break comprises connecting the cell with... The CRISPR/Cas endonuclease is in contact with at least a first gNA and a second gNA, the first gNA containing a target-specific sequence that causes the CRISPR/Cas endonuclease to cleave the target site, and the second gNA containing a target-specific sequence at the 5' end of the template sequence. 96. The method of claim 95, wherein contacting the cells with a CRISPR/Cas endonuclease and sgRNA comprises transfecting the cells with one or more nucleic acid molecules encoding the CRISPR/Cas endonuclease and the sgRNA. 97. The method of any one of claims 87-96, wherein the marker comprises a fluorescent protein operatively linked to a promoter capable of expressing the fluorescent protein in the cell. 98. The method of claim 97, wherein the fluorescent protein comprises GFP, YFP, RFP, CFP, BFP, dsRed, mCherry, or tdTomato. 99. The method of any one of claims 87-98, wherein the mark further includes a selection mark. 100. The method of claim 99, wherein the selection marker is selected from the group consisting of: dihydrofolate reductase (DHFR), glutamine synthase (GS), puromycin acetyltransferase, blastomycin deaminase, histidine dehydrogenase, hygromycin phosphotransferase (hph), bleomycin resistance gene and aminoglycoside phosphotransferase (neomycin resistance). 101. The method of any one of claims 87-100, wherein the cells comprise embryonic stem (ES) cells. 102. The method of any one of claims 87-101, wherein the nucleic acid molecule is a plasmid. 103. A cell comprising the chromosomal rearrangement of any one of claims 87-101. 104. The cell of claim 103, wherein the cell is a mouse ES cell. 105. A transgenic mouse derived from mouse ES cells produced from the cells of claim 103 or 104.