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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细胞类型Cell type	靶位点Target site	靶序列(5′→3′)Target sequence	方向Strand
绵羊胎儿成纤维细胞 Sheep fetal fibroblasts	SOCS2-gRNA1	CCATCTTGGTATTCGATGCG	-
	SOCS2-gRNA2	GATGCGAAGATTAGTTGGTC	-
	SOCS2-gRNA3	TGGGCCTGTCCGCTTATCCT	-
	SOCS2-gRNA4	GCATCGAATACCAAGATGGG	+
	SOCS2-gRNA5	AGATGTGCAAGGATAAGCGG	+
	TBXT-gRNA1	ACGTACTTCCAGCGGTGGTT	-
	TBXT-gRNA2	CGTCGAGGAAAGCTTTTGCA	-
	TBXT-gRNA3	CCTCGACGCAAAGGAAAGGT	+
	TBXT-gRNA4	GAGAGCTGTTGCGATGCGCG	-
	TBXT-gRNA5	GGGTCCAGGCCGGATACGTT	-
绵羊后腿肌细胞 Sheep hind leg myoblasts	MSTN-gRNA1	GAAGACGATGACTACCACGT	+
	MSTN-gRNA2	CGTCGTAACGTGGTAGTCAT	-
	MSTN-gRNA3	CGACGGAAACGGTCATTACC	+
	MSTN-gRNA4	GAGGGTAACGACAGCATCGA	-
	MSTN-gRNA5	CCATGCCCACGGAGTGTGAG	+
	Myf6-gRNA1	CGCCGGTCGGTGGGGGCAGA	-
	Myf6-gRNA2	TTGAGCCGCCGCCTCTCGCG	-
	Myf6-gRNA3	ATGGCGCTCCGCAGAATCTC	-
	Myf6-gRNA4	ATTCATCGGCCTCGAGTAGC	+
	Myf6-gRNA5	TGCGGAGCGCCATTAACTAC	+
	MyoG-gRNA1	CGCAGCGCCATCCAGTACAT	+
	MyoG-gRNA2	GAGGCCCTCAAGAGGAGCAC	+
	MyoG-gRNA3	AAGGGGCACGCGAGCCTCAG	-
	MyoG-gRNA4	GGGTTGAGCAGGGTGCTCCT	-
	MyoG-gRNA5	TCTTCAAGGGGCACGCGAGC	-

靶位点 Target site	打靶载体上游退火引物(5′→3′) hfCas12Max F-oligo	打靶载体下游退火引物(5′→3′) hfCas12Max R-oligo
SOCS2-gRNA1	ACACccatcttggtattcgatgcg	AAAAcgcatcgaataccaagatgg
SOCS2-gRNA2	ACACgatgcgaagattagttggtc	AAAAgaccaactaatcttcgcatc
SOCS2-gRNA3	ACACtgggcctgtccgcttatcct	AAAAaggataagcggacaggccca
SOCS2-gRNA4	ACACgcatcgaataccaagatggg	AAAAcccatcttggtattcgatgc
SOCS2-gRNA5	ACACagatgtgcaaggataagcgg	AAAAccgcttatccttgcacatct
TBXT-gRNA1	ACACacgtacttccagcggtggtt	AAAAaaccaccgctggaagtacgt
TBXT-gRNA2	ACACcgtcgaggaaagcttttgca	AAAAtgcaaaagctttcctcgacg
TBXT-gRNA3	ACACcctcgacgcaaaggaaaggt	AAAAacctttcctttgcgtcgagg
TBXT-gRNA4	ACACgagagctgttgcgatgcgcg	AAAAcgcgcatcgcaacagctctc
TBXT-gRNA5	ACACgggtccaggccggatacgtt	AAAAaacgtatccggcctggaccc
MSTN-gRNA1	ACACgaagacgatgactaccacgt	AAAAacgtggtagtcatcgtcttc
MSTN-gRNA2	ACACcgtcgtaacgtggtagtcat	AAAAatgactaccacgttacgacg
MSTN-gRNA3	ACACcgacggaaacggtcattacc	AAAAggtaatgaccgtttccgtcg
MSTN-gRNA4	ACACgagggtaacgacagcatcga	AAAAtcgatgctgtcgttaccctc
MSTN-gRNA5	ACACccatgcccacggagtgtgag	AAAActcacactccgtgggcatgg
Myf6-gRNA1	ACACcgccggtcggtgggggcaga	AAAAtctgcccccaccgaccggcg
Myf6-gRNA2	ACACttgagccgccgcctctcgcg	AAAAcgcgagaggcggcggctcaa
Myf6-gRNA3	ACACatggcgctccgcagaatctc	AAAAgagattctgcggagcgccat
Myf6-gRNA4	ACACattcatcggcctcgagtagc	AAAAgctactcgaggccgatgaat
Myf6-gRNA5	ACACtgcggagcgccattaactac	AAAAgtagttaatggcgctccgca
MyoG-gRNA1	ACACcgcagcgccatccagtacat	AAAAatgtactggatggcgctgcg
MyoG-gRNA2	ACACgaggccctcaagaggagcac	AAAAgtgctcctcttgagggcctc
MyoG-gRNA3	ACACaaggggcacgcgagcctcag	AAAActgaggctcgcgtgcccctt
MyoG-gRNA4	ACACgggttgagcagggtgctcct	AAAAaggagcaccctgctcaaccc
MyoG-gRNA5	ACACtcttcaaggggcacgcgagc	AAAAgctcgcgtgccccttgaaga

靶位点 Target site	引物F(5′→3′) Primer-F	引物R(5′→3′) Primer-R	产物大小/bp Products size
SOCS2-gRNA1	attaaacggacccacattttca	tttacatagctgcattcggaga	712
SOCS2-gRNA2	attaaacggacccacattttca	gtcttgttggtaaaggcagtcc	614
SOCS2-gRNA3	ggttggtactggggaaatatga	tttacatagctgcattcggaga	531
SOCS2-gRNA4	attaaacggacccacattttca	tttacatagctgcattcggaga	712
SOCS2-gRNA5	ggttggtactggggaaatatga	tttacatagctgcattcggaga	531
TBXT-gRNA1	gatctaggtgagtcctgggaag	acactgagggcttaattgagga	550
TBXT-gRNA2	agcccttgtcagcagtaggtta	agacaggctagtccctgagaca	562
TBXT-gRNA3	agcccttgtcagcagtaggtta	agacaggctagtccctgagaca	562
TBXT-gRNA4	gtggctcgtgggattttagtt	ggcctcatacttggagaaacac	643
TBXT-gRNA5	gatctaggtgagtcctgggaaggagcaccg	tgctgaaggagacaggtgccttcatccag	377
MSTN-gRNA1	ctgtgtaatgcatgcttgtggagac	gaaacagataatgtagcagctttcagtctc	465
MSTN-gRNA2	ctgtgtaatgcatgcttgtggagac	gaaacagataatgtagcagctttcagtctc	465
MSTN-gRNA3	ctgtgtaatgcatgcttgtggagac	gaaacagataatgtagcagctttcagtctc	465
MSTN-gRNA4	aaaggcagaatcaagcctagtg	acagcgatctactaccatgcct	534
MSTN-gRNA5	ctgtgtaatgcatgcttgtggagac	gaaacagataatgtagcagctttcagtctc	465
Myf6-gRNA1	tcctctgtatccagggagtgat	cacctaatcagcctatccgttc	485
Myf6-gRNA2	tcctctgtatccagggagtgat	cacctaatcagcctatccgttc	485
Myf6-gRNA3	tcctctgtatccagggagtgat	cacctaatcagcctatccgttc	485
Myf6-gRNA4	agaaagcaatgctcggagccg	aaggattaggcgaagcagggagg	338
Myf6-gRNA5	tcctctgtatccagggagtgat	cacctaatcagcctatccgttc	485
MyoG-gRNA1	gagacctctccctacttctatcaggaacc	cctttgtcttgtctggaaaggacacgg	672
MyoG-gRNA2	gagacctctccctacttctatcaggaacc	cctttgtcttgtctggaaaggacacgg	672
MyoG-gRNA3	gagacctctccctacttctatcaggaacc	atcgcgctcctcctggttga	429
MyoG-gRNA4	gagacctctccctacttctatcaggaacc	cctttgtcttgtctggaaaggacacgg	672
MyoG-gRNA5	gagacctctccctacttctatcaggaacc	atcgcgctcctcctggttga	429

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