150 Enhancing specificity of gene editing outcomes by using Cas9 variants in swine embryos

CRISPR-Cas9 technology has improved the ability to introduce targeted modifications in cells and embryos in diverse species. Use of the technology allows establishing genetically modified animal models to study human diseases or improve food production. However, one of the main concerns with use of the technology is the possibility of introducing unintended genome modifications (i.e. off-targeting events). Unexpected targeting events elsewhere in the genome are considered a side effect of using gene-editing technology to establish genetically modified animals. The Streptococcus pyogenes Cas9 (SpCas9) exhibits high editing efficacy; however, specificity of the Cas9 nuclease has been questionable, as off-targeting events are known to be introduced. Previously, CRISPR/SpCas9 designed to target swine IGH resulted in an 100% on-target rate, but it also modified loci on AR and RBFOX1 when introduced into fertilized oocytes. Recent developments in CRISPR/Cas9 technology offer several Cas9 variants that were developed to improve gene editing specificity. Here, we employed three high-fidelity SpCas9 variants (eSpCas9, HiFi Cas9, LZ3 Cas9) to examine their efficacy and specificity in pigs by using IGH targeting CRISPR/Cas9 systems as a model. Messenger RNA coding for Cas9 variants mixed with IGH sgRNA were injected into fertilized swine oocytes. Then, IGH, AR, and RBFOX1 regions were amplified from genomic DNA derived from the injected embryos at the blastocyst stage and sent for Sanger sequencing. Injecting the Cas9 variants at 20 ng µL⁻¹ could modify the target gene (IGH) at 100% efficiency, with the excepttion of LZ3 Cas9 (59.1%, n = 22). Importantly, almost no off-target events on AR and RBFOX1 were detected in eSpCas9 (0% for AR and RBFOX1, n = 23), HiFi Cas9 (0% for AR and 4.5% for RBFOX1, n = 22), or LZ3 Cas9 (0% for AR and RBFOX1, n = 22), while 55.6% (AR, n = 18) and 61.1% (RBFOX1, n = 18) off-targeting rate was observed in the SpCas9 group. Frequency of embryos reaching blastocysts on Day 7 post-fertilization was 1.5-fold and 2.5-fold higher in eSpCas9 (33.0%, P < 0.05, Chi-square test) and HiFi Cas9 (49.5%, P < 0.01, Chi-square test) groups, respectively, compared with SpCas9 mRNA (20%) at the same concentration (20 ng µL⁻¹). The growth rate in LZ3 groups was also increased but did not reach statistical significance (31.3%, P > 0.05). In summary, we compared the fidelity of different Cas9 variants to reduce off-target events without compromising on-target success. Our findings indicate eSpCas9 and HiFi Cas9 variants can advance the field of gene editing in livestock models.