Efficient Generation of Knock-Out Sheep by Electroporation of CRISPR-Cas9 Ribonucleoprotein Complex with Dual-sgRNAs.

In mice, naturally occurring and induced mutations in the suppressor of cytokine signaling-2 () gene are associated with a high growth phenotype characterized by rapid post-weaning weight gain and 30-50% heavier mature body weight. In this work, we demonstrate an electroporation-based method of producing knock-out (KO) sheep. Electroporation of dual-guide CRISPR-Cas9 ribonucleoprotein complexes targeting was performed 6 h post-fertilization in sheep zygotes. Fifty-two blastocysts were transferred to 13 estrus-synchronized recipients, yielding five live lambs and one stillborn. These lambs all carried mutations predicted to result in KO. Three carried large deletion alleles which evaded detection in initial PCR screening. Off-target analysis using whole genome sequencing comparing the frequency of mutations in regions within 100 bp of possible sgRNA binding sites (up to 4 bp mismatches) and elsewhere in the genome showed no significant difference when comparing unedited control sheep to edited animals ( = 0.71). In conclusion, electroporation of zygotes with dual-guide CRISPR-Cas9 RNPs was effective at generating knock-out sheep with no substantial off-target activity.