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EP-4735607-A1 - METHODS AND COMPOSITIONS FOR INCREASING HOMOLOGY-DIRECTED REPAIR

EP4735607A1EP 4735607 A1EP4735607 A1EP 4735607A1EP-4735607-A1

Abstract

Provided herein are combinations comprising CRISPR/Cas systems, CtBP-interacting protein, and inhibitor of 53BP1 for use in enhancing homology-directed repair of CRISPR/Cas-mediated cleavage of a target DNA by an exogenous donor nucleic acid. Also provided are methods of using such combinations to make a targeted genetic modification in a cell by homology-directed repair of CRISPR/Cas-mediated cleavage at a target genomic locus in the cell.

Inventors

  • LEE, Wei Ting Chelsea
  • BONETTI, Ciro

Assignees

  • Regeneron Pharmaceuticals, Inc.

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. A method for making a targeted genetic modification by homology- directed repair at a target genomic locus in a cell, comprising administering to the cell: (a) a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) protein or a nucleic acid encoding the Cas protein; (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises one or more adaptor-binding elements to which an adaptor protein can specifically bind, and wherein the guide RNA is capable of forming a complex with the Cas protein and guiding it to a guide RNA target sequence at the target genomic locus; (c) a fusion protein or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises a CtBP-interacting protein (CtIP) fused to the adaptor protein; (d) an inhibitor of 53BP1 (i53) protein or a nucleic acid encoding the i53 protein; and (e) an exogenous donor nucleic acid comprising a 5’ homology arm that hybridizes to a 5’ target sequence at the target genomic locus and a 3’ homology arm that hybridizes to a 3’ target sequence at the target genomic locus, optionally wherein the 5’ homology arm and the 3’ homology arm flank an insert nucleic acid, wherein the Cas protein and the guide RNA form a complex, the Cas protein cleaves the guide RNA target sequence to create a double-strand break, and the exogenous donor nucleic acid recombines with the target genomic locus via homology -directed repair to create the targeted genetic modification.
  2. 2. The method of claim 1, wherein the Cas protein is administered to the cell in the form of a protein, optionally wherein the Cas protein is in a lipid nanoparticle.
  3. 3. The method of claim 1, wherein the nucleic acid encoding the Cas protein is administered to the cell, wherein the nucleic acid encoding the Cas protein comprises an RNA encoding the Cas protein, optionally wherein the RNA encoding the Cas protein is in a lipid nanoparticle.
  4. 4. The method of claim 1, wherein the nucleic acid encoding the Cas protein is administered to the cell, wherein the nucleic acid encoding the Cas protein comprises a DNA encoding the Cas protein, optionally wherein the DNA encoding the Cas protein is in a viral vector, optionally wherein the viral vector is a recombinant adeno-associated virus (AAV) vector.
  5. 5. The method of any one of claims 1-4, wherein the Cas protein is a Cas9 protein.
  6. 6. The method of claim 5, wherein the Cas9 protein is a Streptococcus pyogenes Cas9 protein, a Campylobacter jejuni Cas9 protein, or a Staphylococcus aureus Cas9 protein, optionally wherein the Cas9 protein is the Streptococcus pyogenes Cas9 protein.
  7. 7. The method of any one of claims 1-6, wherein the guide RNA is administered in the form of RNA, optionally wherein the guide RNA is in a lipid nanoparticle.
  8. 8. The method of any one of claims 1-6, wherein the one or more DNAs encoding the guide RNA are administered to the cell, optionally wherein the one or more DNAs encoding the guide RNA are in a viral vector, optionally wherein the viral vector is a recombinant AAV vector.
  9. 9. The method of any one of claims 1-8, wherein the guide RNA comprises two adaptor-binding elements to which the adaptor protein can specifically bind.
  10. 10. The method of claim 9, wherein a first adaptor-binding element is within a first loop of the guide RNA, and a second adaptor-binding element is within a second loop of the guide RNA.
  11. 11. The method of claim 10, wherein the guide RNA is a single guide RNA comprising a CRISPR RNA (crRNA) portion fused to a transactivating CRISPR RNA (tracrRNA) portion, and wherein the first loop is the tetraloop corresponding to residues 13-16 of SEQ ID NO: 11, 13, 15, or 16, and the second loop is the stem loop 2 corresponding to residues 53-56 of SEQ ID NO: 11, 13, 15, or 16.
  12. 12. The method of any one of claims 1-11, wherein the adaptor-binding element comprises the sequence set forth in SEQ ID NO: 19 or 20.
  13. 13. The method of any one of claims 1-12, wherein the guide RNA comprises the sequence set forth in SEQ ID NO: 21, 22, 23, 24, 25, or 26.
  14. 14. The method of any one of claims 1-13, wherein the fusion protein is administered to the cell in the form of a protein, optionally wherein the fusion protein is in a lipid nanoparticle.
  15. 15. The method of any one of claims 1-13, wherein the nucleic acid encoding the fusion protein is administered to the cell, wherein the nucleic acid encoding the fusion protein comprises an RNA encoding the fusion protein, optionally wherein the RNA encoding the fusion protein is in a lipid nanoparticle.
  16. 16. The method of any one of claims 1-13, wherein the nucleic acid encoding the fusion protein is administered to the cell, wherein the nucleic acid encoding the fusion protein comprises a DNA encoding the fusion protein, optionally wherein the DNA encoding the fusion protein is in a viral vector, optionally wherein the viral vector is a recombinant AAV vector.
  17. 17. The method of any one of claims 1-16, wherein the adaptor protein comprises an MS2 coat protein or a functional fragment or variant thereof.
  18. 18. The method of any one of claims 1-17, wherein the adaptor protein comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 32.
  19. 19. The method of any one of claims 1-18, wherein the adaptor protein is encoded by a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 33.
  20. 20. The method of any one of claims 1-19, wherein the CtIP protein comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 34.

Description

METHODS AND COMPOSITIONS FOR INCREASING HOMOLOGY-DIRECTED REPAIR CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of US Application No. 63/511,361, filed June 30, 2023, which is herein incorporated by reference in its entirety for all purposes. REFERENCE TO A SEQUENCE LISTING SUBMITTED AS AN XML FILE VIA EFS WEB [0002] The Sequence Listing written in file 614575SEQLIST.xml is 76,084 bytes, was created on June 27, 2024, and is hereby incorporated by reference. BACKGROUND [0003] The development of CRISPR/Cas technology provides an efficient approach to introduce site-specific modifications in the mammalian genome, offering great potential to study and treat a wide range of genetic diseases. Currently, the most used CRISPR system in genome engineering uses a single-guide RNA (sgRNA) and a CRISPR-associated endonuclease (Cas9), which generate double-stranded breaks (DSBs) at the targeted sequence. The two major DSB repair pathways in mammalian cells are (i) the error-prone non-homologous end joining (NHEJ) and (ii) the faithful homology-directed repair (HDR), which is restricted to the S and G2 phases of the cell cycle and depends on the availability of repair template carrying the modifications to be introduced. Methods to enhance HDR would be useful to provide better and more efficient ways to perform precise genome editing. SUMMARY [0004] Provided herein are combinations comprising CRISPR/Cas systems, CtBP-interacting protein, and inhibitor of 53BP1 for use in enhancing homology-directed repair of CRISPR/Cas- mediated cleavage of a target DNA by an exogenous donor nucleic acid. Also provided are methods of using such combinations to make a targeted genetic modification in a cell by homology-directed repair of CRISPR/Cas-mediated cleavage at a target genomic locus in the cell. [0005] In one aspect, provided are methods for making a targeted genetic modification by homology-directed repair at a target genomic locus in a cell. Some such methods comprise administering to the cell: (a) a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) protein or a nucleic acid encoding the Cas protein; (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises one or more adaptor-binding elements to which an adaptor protein can specifically bind, and wherein the guide RNA is capable of forming a complex with the Cas protein and guiding it to a guide RNA target sequence at the target genomic locus; (c) a fusion protein or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises a CtBP-interacting protein (CtIP) fused to the adaptor protein; (d) an inhibitor of 53BP1 (i53) protein or a nucleic acid encoding the i53 protein; and (e) an exogenous donor nucleic acid comprising a 5’ homology arm that hybridizes to a 5’ target sequence at the target genomic locus and a 3’ homology arm that hybridizes to a 3’ target sequence at the target genomic locus, optionally wherein the 5’ homology arm and the 3’ homology arm flank an insert nucleic acid, wherein the Cas protein and the guide RNA form a complex, the Cas protein cleaves the guide RNA target sequence to create a double-strand break, and the exogenous donor nucleic acid recombines with the target genomic locus via homology- directed repair to create the targeted genetic modification. [0006] In some such methods, the Cas protein is administered to the cell in the form of a protein, optionally wherein the Cas protein is in a lipid nanoparticle. In some such methods, the nucleic acid encoding the Cas protein is administered to the cell, wherein the nucleic acid encoding the Cas protein comprises an RNA encoding the Cas protein, optionally wherein the RNA encoding the Cas protein is in a lipid nanoparticle. In some such methods, the nucleic acid encoding the Cas protein is administered to the cell, wherein the nucleic acid encoding the Cas protein comprises a DNA encoding the Cas protein, optionally wherein the DNA encoding the Cas protein is in a viral vector, optionally wherein the viral vector is a recombinant adeno- associated virus (AAV) vector. In some such methods, the Cas protein is a Cas9 protein. In some such methods, the Cas9 protein is a Streptococcus pyogenes Cas9 protein, a Campylobacter jejuni Cas9 protein, or a Staphylococcus aureus Cas9 protein, optionally wherein the Cas9 protein is the Streptococcus pyogenes Cas9 protein. [0007] In some such methods, the guide RNA is administered in the form of RNA, optionally wherein the guide RNA is in a lipid nanoparticle. In some such methods, the one or more DNAs encoding the guide RNA are administered to the cell, optionally wherein the one or more DNAs encoding the guide RNA are in a viral vector, optionally wherein the viral vector is a recombinant AAV vector. In some such methods, the guide RNA comprises two adaptor-binding elements to which the adaptor protein can specifically bind. In some such me