Methods and compositions for targeted genetic modification using paired guide RNAs

Patent metadata

Field	Value
Publication number	US-10457960-B2
Application number	US-201514948221-A
Country	US
Kind code	B2
Filing date	Nov 20, 2015
Priority date	Nov 21, 2014
Publication date	Oct 29, 2019
Grant date	Oct 29, 2019

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. An in vitro method for making a biallelic modification to a genomic target locus in a genome within a cell, comprising:
(I) introducing into a population of cells:
(a) a Cas protein;
(b) a first guide RNA that hybridizes to a first CRISPR RNA recognition sequence within the genomic target locus;
(c) a second guide RNA that hybridizes to a second CRISPR RNA recognition sequence within the genomic target locus; and
(d) a targeting vector comprising a nucleic acid insert flanked by a 5′ homology arm that hybridizes to a 5′ target sequence within the genomic target locus and a 3′ homology arm that hybridizes to a 3′ target sequence within the genomic target locus;
wherein the genome comprises a pair of first and second homologous chromosomes comprising the genomic target locus; and
wherein the Cas protein cleaves at least one of the first and second CRISPR RNA recognition sequences to generate at least one double-strand break in each of the first and second homologous chromosomes; and

(II) identifying a cell comprising a modified genomic target locus comprising a deletion and/or an insertion, wherein the identifying comprises performing a quantitative modification-of-allele assay and a retention assay,
wherein the modification-of-allele assay comprises:
(a) a gain-of-allele assay to determine a copy number of a region of the nucleic acid insert in a genomic DNA sample from the cell; and/or
(b) a loss-of-allele assay to determine a copy number in the genomic DNA sample of a region of the genomic target locus targeted for deletion, and
wherein the retention assay determines a copy number in the genomic DNA sample of a region of the 5′ target sequence to which the 5′ homology arm hybridizes and/or determines a copy number in the genomic DNA sample of a region of the 3′ target sequence to which the 3′ homology arm hybridizes,
wherein the combination of the modification-of-allele assay and the retention assay distinguishes correct targeted insertion of the nucleic acid insert into the genomic target locus from random transgenic insertions of the nucleic acid insert into genomic locations outside of the genomic target locus and/or distinguishes correct targeted deletions from deletions extending beyond the region of the genomic target locus being targeted for deletion.

2. The method of claim 1 , wherein the retention assay determines the copy number of the region of the 5′ target sequence and determines the copy number of the region of the 3′ target sequence in the genomic DNA sample.

3. The method claim 1 , wherein the nucleic acid insert comprises a selection cassette adjacent to a first homology arm that hybridizes to a first target sequence,
wherein the first homology arm is the 5′ homology arm and the first target sequence is the 5′ target sequence, or wherein the first homology arm is the 3′ homology arm and the first target sequence is the 3′ target sequence,
wherein step (II) comprises the retention assay and the gain-of-allele assay and comprises:
(a) obtaining DNA from the cell;
(b) exposing the DNA of the cell to a probe that binds within the first target sequence, a probe that binds within the nucleic acid insert, and a probe that binds within a reference gene having a known copy number, wherein each probe generates a detectable signal upon binding;
(c) detecting the signals from the binding of each of the probes; and
(d) comparing the signal from the reference gene probe to the signal from the first target sequence probe to determine a copy number for the first target sequence, and comparing the signal from the reference gene probe to the signal from the nucleic acid insert probe to determine a copy number for the nucleic acid insert,
wherein a nucleic acid insert copy number of one or two in the gain-of-allele assay and a first target sequence copy number of two in the retention assay indicates targeted insertion of the nucleic acid insert at the genomic target locus, and
wherein a nucleic acid insert copy number of one or more in the gain-of-allele assay and a first target sequence copy number of three or more in the retention assay indicates a random insertion of the nucleic acid insert at a genomic locus other than the genomic target locus.

4. The method of claim 1 , wherein step (I) further comprises introducing into the population of cells:
(e) a third guide RNA that hybridizes to a third CRISPR RNA recognition sequence within the genomic target locus; and
(f) a fourth guide RNA that hybridizes to a fourth CRISPR RNA recognition sequence within the genomic target locus.

5. The method of claim 4 , wherein:
(a) the first CRISPR RNA recognition sequence and the third CRISPR RNA recognition sequence are separated by about 25 bp to about 1 kb; and
(b) the second CRISPR RNA recognition sequence and the fourth CRISPR RNA recognition sequence are separated by about 25 bp to about 1 kb.

6. The method of claim 4 , wherein the first and third CRISPR RNA recognition sequences are a first pair of CRISPR RNA recognition sequences, and the second and fourth CRISPR RNA recognition sequences are a second pair of CRISPR RNA recognition sequences, wherein the first pair and second pair are separated by about 25 bp to about 100 Mb.

7. The method of claim 1 , wherein introducing both the first and second guide RNAs results in increased biallelic modification efficiency compared to introducing either the first guide RNA or second guide RNA alone.

8. The method of claim 1 , wherein the nucleic acid insert is inserted between the 5′ and 3′ target sequences.

9. The method of claim 1 , wherein the cell is diploid, and the biallelic modification results in homozygosity, compound heterozygosity, or hemizygosity at the genomic target locus.

10. The method of claim 1 , wherein the biallelic modification comprises a deletion between the first and second CRISPR RNA recognition sequences in the first homologous chromosome.

11. The method of claim 10 , wherein the biallelic modification comprises the deletion between the first and second CRISPR RNA recognition sequences in both the first and second homologous chromosomes.

12. The method of claim 11 , wherein the biallelic modification further comprises insertion of the nucleic acid insert between the 5′ and 3′ target sequences in both the first and second homologous chromosomes.

13. The method of claim 10 , wherein the biallelic modification comprises:
(a) the deletion between the first and second CRISPR RNA recognition sequences in both the first and second homologous chromosomes, and insertion of the nucleic acid insert between the 5′ and 3′ target sequences in the first homologous chromosome but not in the second homologous chromosome;
(b) the deletion between the first and second CRISPR RNA recognition sequences in the first homologous chromosome, and disruption of the genomic target locus in the second homologous chromosome, wherein the disruption is from non-homologous end joining (NHEJ)-mediated repair of the at least one double-strand break;
(c) the deletion between the first and second CRISPR RNA recognition sequences in the first homologous chromosome, an insertion of the nucleic acid insert between the 5′ and 3′ target sequences in the first homologous chromosome, and disruption of the genomic target locus in the second homologous chromosome, wherein the disruption is from non-homologous end joining (NHEJ)-mediated repair of the at least one double-strand break; or
(d) the deletion between the first and second CRISPR RNA recognition sequences in the first homologous chromosome, and an insertion of the nucleic acid insert between the 5′ and 3′ target sequences in the first homo

Assignees

• Regeneron Pharma

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

• A01K67/0275 Primary

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

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• A01K2227/105

  Murine · CPC title

• C12N15/102 Primary

  Mutagenizing nucleic acids · CPC title

• C12N15/907 Primary

  in mammalian cells · CPC title