Methods and compositions for targeted genetic modification using paired guide RNAs

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.