Comparison of the efficiency and precision of Base editor and CRISPR/Cas9 for inducing defined point mutation (S395F) in ovine embryos.

Abstract

Cytosine base editors (CBEs) and CRISPR/Cas9-mediated HDR method both have the ability to introduce nucleotide substitution into genomes, which exhibit great potential for improving economically important traits in livestock species. The FecGH mutation (g. C1184T, p. S395F) of growth differentiation factor 9 (GDF9) gene increases prolificacy in Cambridge sheep and Belclare sheep. In the present study, we aimed to compare the efficiency and precision of BE4-Gam and CRISPR/Cas9 systems on generating FecGH mutation in ovine genome. First, the microinjection of BE4-Gam mRNA had no adverse effects on development rate after cleavage, and the efficiencies of total mutants and targeted mutants were 8.9% and 7.1%, respectively. Then, the total mutation and targeted mutation rates were improved from 8.5% to 22.5% (p<.01), and 6.4% to 16.3%, respectively, by adjusting the injection time of BE4-Gam mRNA from 14 to 12hr post-insemination (hpi). Furthermore, CRISPR/Cas9-mediated HDR method introduced the FecGH mutation at the efficiency of 16.1%, which was comparable to BE4-Gam system (16.3%). There was no bystander editing event happened in edited embryos caused by CRISPR/Cas9, but the bystander editing efficiency was as high as 15.0% in BE4-Gam-edited embryos. In summary, our findings demonstrated that CRISPR/Cas9-mediated HDR method was more accurate than BE4-Gam system in introducing FecGH into ovine genome, and highlight the potential of the former strategy to modify economically important trait-associated SNPs.