Introduction of the FecGF mutation in GDF9 gene via CRISPR/Cas9 system with single-stranded oligodeoxynucleotide

文献类型： 外文期刊

第一作者： Xu, Xin

作者： Xu, Xin;Zhang, Xuemei;Peng, Xinrong;Li, Wenrong;Liu, Mingjun;Liu, Chunjie

作者机构：

关键词： CRISPR; Cas9; HDR; GDF9; FecGF; Scr7; Sheep

期刊名称：THERIOGENOLOGY （ 影响因子：2.8； 五年影响因子：2.6 ）

ISSN： 0093-691X

年卷期： 2023 年 197 卷

页码：

收录情况： SCI

摘要： The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system has been a recent focus of breeders owing to its potential to improve economically significant traits of livestock. The introduction of defined point mutations into the ovine genome via CRISPR/Cas9-mediated homology-directed repair has been reported; however, indel and mosaic events observed in genetically modified animals limit the practical application of this system in sheep breeding. The FecGF mutation (g. G1111A, p. V371 M) in the growth differentiation factor 9 (GDF9) gene is strongly associated with litter size in Belclare and Norwegian White Sheep. In the present study, we introduced the FecGF mutation in GDF9 by co-injecting the CRISPR/Cas9 system, single-stranded oligodeoxynucleotide (ssODN), and Scr7 into ovine zygotes. Scr7 at various concentrations (0 mu M, 1 mu M, and 2 mu M) had no adverse effects on embryonic development in vitro. No significant differences in total mutation, point mutation, and indel rates in embryos were observed among groups treated with different concentrations of Scr7. However, the mosaicism rates of embryos from zygotes microinjected with 1 and 2 mu M Scr7 were significantly lower than that for 0 mu M Scr7 (7.7% and 7.5% vs. 19.7%). We successfully obtained lambs with defined nucleotide substitutions by the coinjection of Cas9 mRNA, sgRNA, ssODN, and 1 mu M Scr7 into Altay sheep zygotes. The single nucleotide mutation efficiency was 7.69% (3/39) in newborn lambs, with one mosaic. Our findings provide evidence that Scr7 could improve the specificity of the CRISPR/Cas9 system for the introduction of a defined point mutation in livestock to some extent.(c) 2022 Published by Elsevier Inc.

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