Efficient mutagenesis targeting the gene in mice using a combination of Cas9 protein and dual gRNAs.

We injected mouse zygotes with combinations of Cas9 protein, mRNA, and two gRNAs targeting a single exon of type I interferon receptor () to determine the gene targeting efficiencies. Cas9 protein produced on-target mutations more efficiently than mRNA when each was used with a single gRNA, regardless of which gRNA was used. When mRNA and Cas9 protein were co-injected, the on-target efficiency could reach 97.0% when both gRNAs were used, which was higher than when either gRNA was used alone (61.3% and 75.5%, respectively; P<0.05). Co-injection of Cas9 protein with both gRNAs produced the highest on-target mutation rate of any combination (100.0%). Most on-target mutations were deletions of 2 to 113 nucleotides, and there were few off-target mutations in mutant animals. The expression intensity of IFNAR1 was reduced in heterozygous mice (IF) and almost or completely absent in homozygous null mice compared with that in wild-type mice (IF and Western blot). When both gRNAs targeting were used simultaneously with two gRNAs targeting , the on-target editing efficiency on each gene was 96.8% and 85.5%, respectively. Co-injection of dual gRNAs and Cas9 protein is an efficient approach for knockout and multi-gene editing in mice and may be applied in other animal models and breeding livestock.