Efficient Generation ofSOCS2Knock-out Sheep by Electroporation of CRISPR-Cas9 Ribonucleoprotein Complex with Dual-sgRNAs

Abstract

AbstractKnock-out (KO) sheep were produced using CRISPR-Cas9 ribonucleoprotein complexes in zygotes targeting an 85 bp section of the first exon of the Suppressor of Cytokine Signalling-2 (SOCS2) gene. Electroporation was performed 6 hours post-fertilization with dual-guide CRISPR-Cas9 ribonucleoproteins (RNPs). Fifty-two blastocysts were transferred to 13 estrus-synchronized recipients, yielding five live lambs and one stillborn. These lambs were all compound heterozygotes with mutations predicted to result inSOCS2KO. Three lambs carried large deletion alleles (259 bp, 1694 bp, and 2127 bp) that evaded initial detection via initial PCR screening. Off-target analysis identified a small number of mutations which may have been the result of off-target activity in regions with some homology to the guides, but notably such mutations were also observed in unedited controls. Further, we observed several orders of magnitude more mutations outside of these regions of homology in both edited animals and controls. Western blot and RT-PCR analysis of cell lines from SOCS2 KO lambs showed trace levels ofSOCS2mRNA and SOCS2 protein. In conclusion, combining IVF and electroporation of dual-guide CRISPR-Cas9 RNPs was effective at generating KO sheep.