Wang Yu, Bi Dengfeng, Qin Guosong, Song Ruigao, Yao Jing, Cao Chunwei, Zheng Qiantao, Hou Naipeng, Wang Yanfang, Zhao Jianguo
State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China.
State Key Laboratory of Stem cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
Front Genet. 2020 Nov 16;11:592623. doi: 10.3389/fgene.2020.592623. eCollection 2020.
Pig is an important agricultural economic animal, providing large amount of meat products. With the development of functional genomics and bioinformatics, lots of genes and functional single nucleotide polymorphisms (SNPs) related to disease resistance and (or) economic traits in pigs have been identified, which provides the targets for genetic improvement by genome editing. Base editors (BEs), combining Cas9 nickase and cytidine or adenine deaminase, achieve all four possible transition mutations (C-to-T, A-to-G, T-to-C, and G-to-A) efficiently and accurately without double strand breaks (DSBs) under the protospacer adjacent motif (PAM) sequence of NGG. However, the NGG PAM in canonical CRISPR-Cas9 can only cover approximately 8.27% in the whole genome which limits its broad application. In the current study, hA3A-BE3-NG system was constructed with the fusion of SpCas9-NG variant and hA3A-BE3 to create C-to-T conversion at NGN PAM sites efficiently. The editing efficiency and scope of hA3A-BE3-NG were confirmed in HEK293T cells and porcine fetal fibroblast (PFF) cells. Results showed that the efficiency of hA3A-BE3-NG was much higher than that of hA3A-BE3 on NGH (H = A, C, or T) PAM sites (21.27 vs. 2.81% at average). Further, nonsense and missense mutations were introduced efficiently and precisely hA3A-BE3-NG in multiple pig economic trait-related genes (, , , and ) in PFF cells by one transfection. The current work indicates the potential applications of hA3A-BE3-NG for pyramid breeding studies in livestock.
猪是重要的农业经济动物，能提供大量肉类产品。随着功能基因组学和生物信息学的发展，已鉴定出许多与猪抗病性和（或）经济性状相关的基因及功能性单核苷酸多态性（SNP），这为通过基因组编辑进行遗传改良提供了靶点。碱基编辑器（BE）将Cas9切口酶与胞嘧啶或腺嘌呤脱氨酶结合，在NGG的原间隔序列临近基序（PAM）序列下高效且准确地实现所有四种可能的转换突变（C到T、A到G、T到C和G到A），而不会产生双链断裂（DSB）。然而，经典CRISPR-Cas9中的NGG PAM在整个基因组中仅覆盖约8.27%，这限制了其广泛应用。在本研究中，构建了hA3A-BE3-NG系统，将SpCas9-NG变体与hA3A-BE3融合，以在NGN PAM位点高效实现C到T的转换。在人胚肾293T细胞和猪胎儿成纤维细胞（PFF）中证实了hA3A-BE3-NG的编辑效率和范围。结果表明，hA3A-BE3-NG在NGH（H = A、C或T）PAM位点的效率远高于hA3A-BE3（平均分别为21.27%和2.81%）。此外，通过一次转染，hA3A-BE3-NG在PFF细胞中的多个猪经济性状相关基因（、、、和）中高效且精确地引入了无义突变和错义突变。目前的工作表明hA3A-BE3-NG在牲畜金字塔育种研究中的潜在应用。
