151 Assessing the efficiency of cytosine base editors targeting MYO7A in bovine embryos

Owing to its high editing efficiency and simplicity, CRISPR/Cas9 technology is the most prevalent gene editing strategy. CRISPR/Cas9 introduces double-strand breaks, which can lead to DNA repair mechanisms and non-homologous end joining. These outcomes result in random insertions or deletions (indels) and the potential creation of a premature stop codon, leading to a nonfunctional protein. However, triplet indel mutations could lead to the loss or change of one or more amino acids, resulting in a hypomorphic phenotype and partial functionality. Cytosine base editors (CBEs) offer a solution by enabling precise C-to-T (G-to-A) conversions without double-strand breaks, wherein a premature stop codon could be introduced, and thus without the associated risk of a hypomorphic phenotype. Currently, little information exists regarding the efficiency of CBEs in modifying the germline in non-rodent species. In this study, we compared the C-to-T conversion efficiency in targeting exon 4 of the MYO7A gene in bovine embryos using a standard base editor (BE3) and an engineered base editor (eBE3-Y130F) that includes a uracil glycosylase inhibitor (UGI) and a modified cytidine deaminase. A single guide (sg)RNA was designed to disrupt MYO7A, a gene that gives rise to congenital deafness and progressive blindness when mutated in humans (Usher Syndrome Type 1b). To validate the efficiency of the sgRNA, Cas9 mRNA was introduced into 95 bovine zygotes along with the sgRNA. Five blastocysts and three arrested embryos were analyzed to confirm the sgRNA efficiency. The result was 100% indel mutations in all embryos, as determined by Sanger sequencing. The high-efficiency sgRNA was then injected with BE3 or eBE3-Y130F into bovine zygotes. A total of 95 bovine zygotes received BE3, which resulted in three blastocysts. To determine editing efficiency, three blastocysts and two arrested embryos were analyzed for editing. A total of 171 bovine zygotes received eBE3-Y130F with sgRNA, resulting in the development of 15 blastocysts (an 8.77% blastocyst formation rate). For genotyping, 15 blastocysts and an additional four arrested embryos were analyzed. The region flanking the sgRNA binding site was amplified and sequenced from the injected embryos. Strikingly, the BE3 and eBE3-Y130F editors generated C-to-T conversions at 100% efficiency. Results from these studies revealed that (1) C-to-T conversions were detected outside of the sgRNA target site, which were nucleotides 21 bp, 22 bp, and 27 bp distal from the PAM sequence; (2) the BE3 editing window encompassed the third to eighth bases within the sgRNA target sequence, and the editing window of eBE3-Y130F was fifth to eigth base pair of guide binding sequence, both relative to the PAM sequence; and (3) eBE3-Y130F resulted in less homozygous edits within the editing window compared with BE3. In contrast to previous studies using base editors in mouse embryos, our results demonstrated that BE3 showed an enhanced C-to-T conversion rate in the bovine MYO7A gene and a broader conversion window compared with eBE3-Y130F. Future studies will assess BE3 and eBE3-Y130F efficiencies on different target genes in bovine embryos.