小分子药物对CRISPR/Cas12i介导的两种绵羊原代细胞基因敲入效率的影响

doi:10.11843/j.issn.0366-6964.2025.12.015

摘要/Abstract

摘要： 旨在以绵羊原代细胞为材料，验证CRISPR/Cas12i系统介导的以ssODN为供体进行HDR精准修复的基因编辑效率，以及5种小分子药物对Indels效率和敲入效率的影响。本研究以两种绵羊原代细胞为试验对象，验证CRISPR/Cas12i系统在25个靶基因位点的基因编辑效率；然后选取绵羊胎儿成纤维细胞的3个靶点(SOCS2-gRNA1、SOCS2-gRNA3和TBXT-gRNA5)，绵羊后腿肌细胞的5个靶点(MSTN-gRNA2、MSTN-gRNA5、Myf6-gRNA4、Myf6-gRNA5和MyoG-gRNA4)检测5种小分子药物(SCR7、Nocodazole、RS-1、Vorinostat及Entinostat)对各靶点Indels效率的影响，最后在MSTN-gRNA2靶点检测5种小分子药物对CRISPR/Cas12i 系统介导的以ssODN为供体进行HDR精准修复的基因敲入效率的影响。本研究验证了CRISPR/Cas12i系统在两种绵羊原代细胞的25个不同靶点均具有高基因编辑活性，添加小分子药物处理后，SCR7会降低绵羊原代细胞各靶点Indels效率，而RS-1、Entinostat和Vorinostat处理后对各位点Indels效率均有提升效果，Nocodazole对两种细胞作用效果不一致。在添加5种小分子药物处理后，MSTN-gRNA2位点敲入效率均有不同程度提高，其中2 μmol·L^-1 Vorinostat和4 μmol·L^-1 Entinostat处理组提升效果最高，敲入效率分别为7.67%和7.73%。该试验为CRISPR/Cas12i基因编辑系统的应用与推广提供了借鉴，为创制具有优良性状的基因编辑绵羊提供了新思路，为加速精准分子育种工作提供了技术支撑。

关键词: CPRISPR/Cas12i, 同源重组, 绵羊, Indels, 基因敲入

Abstract: Using primary ovine cells as materials, this study aimed to verify the gene editing efficiency of precise repair through homology-directed repair (HDR) with single-stranded oligodeoxynucleotide (ssODN) as the donor mediated by the CRISPR/Cas12i system, and to explore the effects of small molecule drugs on the efficiency of insertions and deletions (Indels) and the knock-in efficiency. In this study, two types of primary ovine cells were used as experimental subjects to verify the gene editing efficiency of the CRISPR/Cas12i system at 25 target gene loci. Then, 3 target sites (SOCS2-gRNA1, SOCS2-gRNA3, and TBXT-gRNA5) in ovine fetal fibroblasts and 5 target sites (MSTN-gRNA2, MSTN-gRNA5, Myf6-gRNA4, Myf6-gRNA5 and MyoG-gRNA4) in ovine hindlimb muscle cells were selected to detect the effects of 5 small molecule drugs (SCR7, Nocodazole, RS-1, Vorinostat, and Entinostat) on the Indel efficiency at each target site. Finally, the effect of the 5 small molecule drugs on the gene knock-in efficiency of precise repair through HDR with ssODN as the donor mediated by the CRISPR/Cas12i system was detected at the MSTN-gRNA2 target site. This study verified that the CRISPR/Cas12i system had high gene editing activity at 25 different target sites in the two types of primary ovine cells. After treatment with small molecule drugs, SCR7 decreased the Indels efficiency at each target site in primary ovine cells, while treatment with RS-1, Entinostat, and Vorinostat increased the Indels efficiency at each site. Nocodazole had inconsistent effects on the two types of cells. After treatment with the 5 small molecule drugs, the knock-in efficiency at the MSTN-gRNA2 site was increased to varying degrees. Among them, the treatment groups with 2 μmol·L^-1 Vorinostat and 4 μmol·L^-1 Entinostat had the highest enhancement effects, with the knock-in efficiencies being 7.67% and 7.73%, respectively. This experiment provides a reference for the application and promotion of the CRISPR/Cas12i gene editing system, offers new ideas for the creation of gene-edited sheep with excellent traits, and provides technical support for accelerating the work of precise molecular breeding.

Key words: CRISPR/Cas12i, homologous recombination, sheep, Indels, gene knock-in

中图分类号:

S826.2

苏大崴, 陈秋崇, 苗洱钰, 张骐镜, 陈哲, 王小龙, 徐坤. 小分子药物对CRISPR/Cas12i介导的两种绵羊原代细胞基因敲入效率的影响[J]. 畜牧兽医学报, 2025, 56(12): 6104-6115.

SU Dawei, CHEN Qiuchong, MIAO Eryu, ZHANG Qijing, CHEN Zhe, WANG Xiaolong, XU Kun. The Effect of Small Molecule Drugs on Gene Knock-in Efficiency Mediated by the CRISPR/Cas12i System in Primary Ovine Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6104-6115.

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