337. CRISPR/Cas9 Mediated Highly Efficient Genome Engineering in Mouse Embryos

Abstract

CRISPR/Cas9 is an RNA-based adaptive immune system used by bacteria and archaea to cleave foreign nucleic acids (e.g. bacteriophage DNA). This system has been adapted as a tool to edit mammalian genes with high efficiency and specificity. Here, we describe our use of the CRISPR/Cas9 system to achieve targeted gene knock outs and targeted gene repair in one cell stage mouse embryos. We have efficiently mutated tyrosinase (a coat color gene), p53 (a tumor suppressor gene), and Cacna1a (a voltage-gated ion channel gene) in mouse embryos by injection of Cas9 mRNA plus either a single or paired guide RNAs (sgRNAs). Single sgRNAs typically induced deletions of 1 bp to several hundred bp at their target site whereas paired sgRNAs generated localized deletions at high efficiency. For gene repair, we used 700 bp to 2 kb donor DNAs and a single sgRNA to promote gene correction by homology-mediated repair. Our experimental data revealed 8 to 18% efficiency of gene correction. When we used two sgRNAs to target a single exon, all of the newborn mice from one experiment showed targeted therapy for an inherited genetic disorder, albinism. No one has previously published a protocol that has achieved gene repair with this level of effectiveness. Our results confirm that the CRISPR/Cas9 system can be used for efficient gene targeting and gene repair in vivo.