Enhancing the specificity of gene editing outcomes by using Cas9 variants in porcine embryos.

The CRISPR/Cas9 technology has improved the ability to introduce targeted modifications in cells and embryos in diverse species. The use of this technology enables the establishment of genetically modified livestock models to study human diseases or improve food production. However, one of the main concerns with employing this technology is the possibility of introducing unintended genome modifications induced by the Streptococcus pyogenes Cas9 (SpCas9), a commonly used Cas9 protein. Recent advancements in CRISPR/Cas9 technology offer Cas9 variants that are designed to improve gene editing specificity. Here, three high-fidelity SpCas9 variants (eSpCas9, HiFi Cas9, and LZ3 Cas9) were employed to examine their efficacy and specificity in pig embryos. To introduce targeted modifications, mRNA coding for each Cas9 variant was mixed with IGH single guide RNA (sgRNA) and were injected into fertilized pig zygotes. The frequency of on- and off-targeting was calculated by amplifying IGH, AR, and RBFOX1 regions from genomic DNA derived from the injected embryos at the blastocyst stage and sent for Sanger sequencing. The sgRNA targeting IGH locus resulted in a 100% on-target editing rate using SpCas9. However, SpCas9 introduced off-targeting events in AR and RBFOX1 at a high frequency (> 60%) in embryos. Injecting each Cas9 variant at 20 ng/µl could modify the target gene (IGH) at 100% efficiency except for LZ3 Cas9 (59.1%). Importantly, off-target events on AR and RBFOX1 were not detected in any Cas9 variant groups. Gradually reducing the concentration of Cas9 mRNAs lowered the efficacy of on-targeting in all groups; however, the reduction was more dramatic in HiFi Cas9 and LZ3 Cas9 injected embryos. No embryonic toxicity was identified in embryo injected with Cas9 variants and more embryos reached blastocyst stage when injected with either eSpCas9 or HiFiCas9 mRNA. In vivo competency of embryos receiving eSpCas9 was examined by embryo transfer and fetuses recovered from a pregnant sow presented 100% on-target editing efficiency without any detectable off-target events. In summary, among the Cas9 variants examined, eSpCas9 presented the highest specificity with no detectable off-target events and supported the development of gene-edited fetuses. Our findings indicate that the use of Cas9 variants can advance the field of gene editing in livestock models.