Editing Efficiency of Four Single Base Editors in Sheep (Ovis aries) and Goat (Capra hircus) Fibroblasts
SUN Jia-Yuan, SUN Ke-Xin, DING Yi-Ge, ZHOU Shi-Wei, GAO Ya-Wei, CHEN Yu-Lin, WANG Xiao-Long*
College of Animal Science and Technology/Shaanxi Key Laboratory of Animal Genetic Breeding and Reproduction, Northwest A&F
University, Yangling 712100, China
Abstract：As a new type of CRISPR-derived tool, the base editors have the advantages of accuracy, efficiency, and low off-target, and have been used in many organisms. Single base editors are mainly divided into 2 types-adenine base editor (ABEs) and cytosine base editor (CBE), which can realize single base mutation of A:G and C:T, respectively. The base editor has been continuously modified and has been developed with multiple versions. The Booroola fecundity (FecB) gene is the major gene of sheep (Ovis aries) prolificacy. FecB gene is derived from A746G site mutation of bone morphogenetic protein receptor 1B (BMPR1B) of Booroola sheep, resulting in 249 amino acids encoded being converted from glutamine (Q) to arginine (R), further increasing ovulation and lambing. The fibroblast growth factor 5 (FGF5) gene can regulate the hair follicle cycle, and C-T base mutation on FGF5 gene can significantly affect the length of cashmere fiber, which is crucial for improving cashmere yield of cashmere goat (Capra hircus). In order to explore the editing efficiency of different base editors in sheep and goat, the Booroola fecundity (FecB) of Tan sheep and the fibroblast growth factor 5 (FGF5) of Shaanbei white cashmere goat were selected. By constructing sgRNA-U6 vector, cell culture, transfection, drug screening, Four single base editors, xCas9-ABE (adenine base editor), ABEmax4, xCas9-BE4 and BE4max, were used to perform single base fixed-point editing on Tan sheep and Shaanbei white cashmere goat fetal fibroblasts. The editing efficiency of xCas9-ABE, ABEmax4, xCas9 -BE4 and BE4max in Tan sheep and Shaanbei white cashmere goat were determined. The results showed that in sheep fibroblasts, the editing efficiency of ABEmax4 was 46.15%, and the editing efficiency of xCas9-ABE was 38.46%, which was 27.4% and 19.71% higher than that of ordinary editor ABE7.10. On goat fibroblasts, the editing efficiency of BE4max It was 92.86%, which was 56.5% higher than the ordinary editor BE3, but no editing is generated by xCas9-BE4. In summary, the base editing application of sheep fetal fibroblasts, the optimal base editors of ABEmax4 and BE4max were obtained. The study screened the optimal base editor in the application of base editing in sheep fetal fibroblasts, which proved the feasibility of efficient site-specific editing of sheep genome, and also provides technical support for the application of base editor in gene editing of large mammals.
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