Generation of knockout mice by Cpf1-mediated gene targeting

VOLUME 34 NUMBER 8 AUGUST 2016 NATURE BIOTECHNOLOGY target sites by four mismatches. We also electroporated an AsCpf1 RNP targeting Tyr, the gene encoding tyrosinase, into mouse embryos and observed mutations in 4 out of 12 blastocysts (33%) (Supplementary Fig. 6). Taken together, these results show that Cas9 and Cpf1 RNPs can be delivered efficiently by electroporation into animal embryos, resulting in high mutation frequencies. We next transplanted mouse embryos after Cpf1 microinjection or electroporation into surrogate mothers and obtained mice with targeted mutations in Foxn1 (Fig. 1d and Supplementary Fig. 7a) or Tyr (Supplementary Table 2 and Supplementary Fig. 8a). Three out of seven mice carried mutations at the Cpf1 cleavage site in the Foxn1 gene. One Tyr mutant mouse showed a partial coat color change, consistent with its mosaic genotype (Supplementary Fig. 8b). To investigate whether Cpf1 had offtarget effects, we performed whole genome sequencing using genomic DNA isolated from one Foxn1 mutant mouse and its wildtype sibling (Supplementary Note). The sequence analysis showed that no off-target mutations were introduced at homologous sites with up to 7-nucleotide mismatches. Notably, the mutant allele in a female Foxn1 mutant mouse was transmitted to embryos (Supplementary Fig. 7b,c). In summary, our results show that electroporation of AsCpf1 RNPs resulted in efficient and specific genome editing in mouse embryos. RNPs7 are as effective as mRNA8 or plasmids9, but are degraded rapidly by endogenous proteases and RNases in cells, and have been previously shown to reduce off-target effects9 and mosaicism10. Unlike microinjection, electroporation is easy to carry out, fast, and scalable. Up to 50 embryos can be electroporated simultaneously. We propose that electroporation of Cpf1 RNPs is a potentially useful new method for genome editing in animals.