Methods and compositions for targeted genetic modification using paired guide RNAs

Patent metadata

Field	Value
Publication number	US-11697828-B2
Application number	US-201916572124-A
Country	US
Kind code	B2
Filing date	Sep 16, 2019
Priority date	Nov 21, 2014
Publication date	Jul 11, 2023
Grant date	Jul 11, 2023

Abstract

Compositions and methods are provided for creating and promoting biallelic targeted modifications to genomes within cells and for producing non-human animals comprising the modified genomes. Also provided are compositions and methods for modifying a genome within a cell that is heterozygous for an allele to become homozygous for that allele. The methods make use of Cas proteins and two or more guide RNAs that target different locations within the same genomic target locus. Also provided are methods of identifying cells with modified genomes.

First claim

We claim:

1. A method for modifying a genome within a mammalian cell in vitro that is heterozygous for a first allele, comprising:
(I) contacting the genome with:
(a) a Cas protein;
(b) a first guide RNA, wherein the first guide RNA comprises a first tracrRNA and a first CRISPR RNA that hybridizes to a first non-allele-specific CRISPR RNA recognition sequence, wherein the cell comprises a chromosome pair of first and second homologous chromosomes comprising a genomic locus, wherein the first allele is at the genomic locus on the first homologous chromosome, and the CRISPR RNA recognition sequence is centromeric to the genomic locus on the second homologous chromosome; and
(c) a second guide RNA, wherein the second guide RNA comprises a second tracrRNA and a second CRISPR RNA that hybridizes to a second non-allele-specific CRISPR RNA recognition sequence centromeric to the genomic locus on the second homologous chromosome,
wherein the first allele is at least 100 kb from the first CRISPR RNA recognition sequence,
wherein the Cas protein and the first guide RNA do not naturally occur together,
wherein the Cas protein is a Cas9 protein and has nuclease activity on both strands of double-stranded DNA, and
wherein the Cas protein cleaves the first CRISPR RNA recognition sequence on the second homologous chromosome to generate a double-strand break and the cell is modified to become homozygous for the first allele; and

(II) identifying a modified cell that is homozygous for the first allele.

2. The method of claim 1 , wherein step (I) comprises introducing into the cell: (a) the Cas protein or a nucleic acid encoding the Cas protein; and (b) the first guide RNA or a DNA encoding the first guide RNA.

3. The method of claim 2 , wherein step (I) comprises introducing into the cell the nucleic acid encoding the Cas protein and introducing into the cell the first guide RNA, wherein the nucleic acid encoding the Cas protein is an RNA.

4. The method of claim 2 , wherein step (I) comprises introducing into the cell the nucleic acid encoding the Cas protein and the DNA encoding the first guide RNA, wherein the nucleic acid encoding the Cas protein is a DNA.

5. The method of claim 2 , wherein the Cas protein and the first guide RNA are introduced into the cell as a first protein-RNA complex.

6. The method of claim 4 , wherein the first CRISPR RNA and the first tracrRNA are separate RNA molecules, and wherein:
(1) the DNA encoding the Cas protein is operably linked to a first promoter in a first expression construct;
(2) the DNA encoding the first CRISPR RNA is operably linked to a second promoter in a second expression construct; and
(3) the DNA encoding the first tracrRNA is operably linked to a third promoter in a third expression construct;
wherein the first, second, and third promoters are active in the cell.

7. The method of claim 6 , wherein the first, second, and third expression constructs are components of a single nucleic acid molecule.

8. The method of claim 4 , wherein:
(1) the DNA encoding the Cas protein is operably linked to a first promoter in a first expression construct; and/or
(2) the DNA encoding the first guide RNA is operably linked to a second promoter in a second expression construct,
wherein the first and second promoters are active in the cell.

9. The method of claim 8 , wherein the first and second expression constructs are components of a single nucleic acid molecule.

10. The method of claim 1 , wherein the double-strand break is repaired in the cell, and wherein repair of the double-strand break results in loss of heterozygosity that occurs telomeric of the double-strand break.

11. The method of claim 1 , wherein step (I) further comprises introducing into the cell the second guide RNA or a DNA encoding the second guide RNA.

12. The method of claim 1 , wherein the Cas protein cleaves the first CRISPR RNA recognition sequence and the second CRISPR RNA recognition sequence.

13. The method of claim 1 , wherein the first CRISPR RNA recognition sequence is from 100 bp to 1 kb, 1 kb to 10 kb, 10 kb to 100 kb, 100 kb to 1 Mb, 1 Mb to 10 Mb, 10 Mb to 20 Mb, 20 Mb to 30 Mb, 30 Mb to 40 Mb, 40 Mb to 50 Mb, 50 Mb to 60 Mb, 60 Mb to 70 Mb, 70 Mb to 80 Mb, 80 Mb to 90 Mb, or 90 Mb to 100 Mb from the centromere.

14. The method of claim 1 , wherein the first allele is from 100 kb to 1 Mb, 1 Mb to 10 Mb, 10 Mb to 20 Mb, 20 Mb to 30 Mb, 30 Mb to 40 Mb, 40 Mb to 50 Mb, 50 Mb to 60 Mb, 60 Mb to 70 Mb 70 Mb to 80 Mb, 80 Mb to 90 Mb, or 90 Mb to 100 Mb from the first CRISPR RNA recognition sequence.

15. The method of claim 1 , wherein the first allele is at least 1 Mb, at least 10 Mb, at least 20 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, or at least 100 Mb from the first CRISPR RNA recognition sequence.

16. The method of claim 1 , wherein a region of the second homologous chromosome is modified to become homozygous for the first allele by loss of heterozygosity, wherein the region of the second homologous chromosome being replaced by loss of heterozygosity is from 100 bp to 1 kb, 1 kb to 10 kb, 10 to 100 kb, 100 kb to 1 Mb, 1 Mb to 10 Mb, 10 Mb to 20 Mb, 20 Mb to 30 Mb, 30 Mb to 40 Mb, 40 Mb to 50 Mb, 50 Mb to 60 Mb, 60 Mb to 70 Mb, 70 Mb to 80 Mb, 80 Mb to 90 Mb, or 90 Mb to 100 Mb.

17. The method of claim 1 , wherein a region of the second homologous chromosome is modified to become homozygous for the first allele by loss of heterozygosity, wherein the region of the second homologous chromosome being replaced by loss of heterozygosity is at least 100 bp, at least 1 kb, at least 10 kb, at least 100 kb, at least 1 Mb, at least 10 Mb, at least 20 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, or at least 100 Mb.

18. The method of claim 1 , wherein:
(1) the first allele comprises a mutation; or
(2) the first allele is a wild type allele, and the genomic locus on the second homologous chromosome comprises a mutation.

19. The method of claim 18 , wherein the first allele comprises the mutation, wherein the mutation is a targeted modification.

20. The method of claim 1 , wherein the cell is a human cell, a non-human cell, a rodent cell, a mouse cell, a rat cell, a pluripotent cell, a non-pluripotent cell, a non-human pluripotent cell, a human pluripotent cell, a rodent pluripotent cell, a mouse pluripotent cell, a rat pluripotent cell, a mouse embryonic stem (ES) cell, a rat ES cell, a non-human mammalian ES cell, a human adult stem cell, a developmentally restricted human progenitor cell, a human induced pluripotent stem (iPS) cell, a non-human mammalian one-cell stage embryo, or a mouse one-cell stage embryo.

21. The method of claim 20 , wherein the cell is the human iPS cell.

22. The method of claim 20 , wherein the cell is the mouse ES cell or the rat ES cell.

23. The method of claim 22 , wherein the cell is the mouse ES cell.

24. The method of claim 1 , wherein the first CRISPR RNA and the first tracrRNA in the first guide RNA are fused together.

25. The method of claim 1 , wherein the first CRISPR RNA and the first tracrRNA are separate RNA molecules.

26. The method of claim 1 , wherein the cell has been modified to decrease non-homologous end joining (NHEJ) and/or to increase gene conversion or homology-directed repair (HDR).

Assignees

• Regeneron Pharma
• Regeneran Pharmaceuticals Inc

Inventors

• Frendewey David
• Lai Ka-Man Venus
• Auerbach Wojtek
• Droguett Gustavo
• Gagliardi Anthony
• Valenzuela David M
• Voronina Vera
• Macdonald Lynn
• Murphy Andrew J
• Yancopoulos George D

Classifications

• A01K2227/105

  Murine · CPC title

• A01K67/0275 Primary

  Genetically modified vertebrates, e.g. transgenic · CPC title

• C12N15/102 Primary

  Mutagenizing nucleic acids · CPC title

• C12N15/907 Primary

  in mammalian cells · CPC title

• C12N15/8509

  for producing genetically modified animals, e.g. transgenic · CPC title