基于CRISPR-Cas12i技术构建XIST基因敲除的华西牛成纤维细胞系

doi:10.11843/j.issn.0366-6964.2025.11.010

摘要/Abstract

摘要：

旨在利用Cas12i^HIFI基因编辑技术获得敲除XIST基因华西牛成纤维细胞系。本研究使用健康成年种公牛22284#的耳缘成纤维细胞作为供体，针对牛XIST基因设计多组sgRNA，验证效率后选取6对高效的sgRNA组成3个方案进行试验，每个方案收集15份九十六孔板细胞，通过电转染方式介导基因编辑，最后使用突变细胞验证出使用的sgRNA没有出现脱靶情况。结果表明，成功构建了XIST敲除的华西牛成纤维细胞模型。试验共获得5株纯合突变细胞系及1株杂合细胞系，验证了Cas12i在高GC含量重复序列中的高效编辑能力。该研究为解析牛XCI机制及提高克隆胚胎发育效率提供了重要的试验材料和理论依据。

关键词: X染色体失活, XIST基因, 重复序列, 基因编辑, CRISPR/Cas12i

Abstract:

This study aimed to generate XIST-knockout fibroblast cell lines in Chinese Huaxi cattle using CRISPR-Cas12i^HIFI-mediated gene editing. Ear-edge fibroblast cells from a healthy adult bull (No. 22284#) were used as donors. Multiple sgRNAs were designed targeting the bovine XIST gene, and 6 highly efficient sgRNAs were selected and grouped into 3 editing strategies. A total of 15 ninety-six-well plates of cells were collected per strategy and subjected to electroporation. Off-target analysis confirmed the specificity of the selected sgRNAs. As a result, we successfully established XIST-knockout fibroblast models, obtaining five homozygous and one heterozygous mutant cell line. This results demonstrates the high efficiency of Cas12i in editing GC-rich repetitive sequences. Our work provides essential experimental material and theoretical basis for elucidating the mechanism of X-chromosome inactivation (XCI) and improving the developmental efficiency of cloned bovine embryos.

Key words: X chromosome inactivation, XIST gene, repetitive sequences, gene editing, CRISPR/Cas12i

中图分类号:

S823.2

张留哲, 赵佳男, 张丽琼, 张裕荣, 唐露, 李俊良, 郭慧慧. 基于CRISPR-Cas12i技术构建XIST基因敲除的华西牛成纤维细胞系[J]. 畜牧兽医学报, 2025, 56(11): 5464-5474.

ZHANG Liuzhe, ZHAO Jianan, ZHANG Liqiong, ZHANG Yurong, TANG Lu, LI Junliang, GUO Huihui. Construction of XIST Gene Knockout Fibroblast Cell Line from Huaxi Cattle Using CRISPR-Cas12i Technology[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5464-5474.

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sgRNA名称 sgRNA name	sgRNA序列 sgRNA sequence
1-1	acggTTAAAGCGCTGCACTTTGCT	aaaaAGCAAAGTGCAGCGCTTTAA
1-2	acggACAGACACGAACCCATTGAA	aaaaTTCAATGGGTTCGTGTCTGT
1-3	acggAAGTCATGGCTCCTGGACTA	aaaaTAGTCCAGGAGCCATGACTT
1-4	acggCTGGAACATTTTCCAGACCC	aaaaGGGTCTGGAAAATGTTCCAG
1-5	acggCCATACTAGTCACTTAAGGC	aaaaGCCTTAAGTGACTAGTATGG
1-6	acggCTAGTGTTCGATTTCAGCCT	aaaaAGGCTGAAATCGAACACTAG
1-7	acggCTCAAGAGGAACACCTACCC	aaaaGGGTAGGTGTTCCTCTTGAG
1-8	acggATGGGTTTTCATATTTGGGT	aaaaACCCAAATATGAAAACCCAT
1-9	acggATCTGATACCAATGCCCTTT	aaaaAAAGGGCATTGGTATCAGAT
1-10	acggTCAGGCAGGGGCTCTCATAT	aaaaATATGAGAGCCCCTGCCTGA
2-1	acggATTAAAATGGGCAGTGAGAG	aaaaCTCTCACTGCCCATTTTAAT
2-2	acggTATAGAACTTGGAGATCGTG	aaaaCACGATCTCCAAGTTCTATA
2-3	acggCTCACAGCTTGTTTCCCCCA	aaaaTGGGGGAAACAAGCTGTGAG
2-4	acggCCTCCAGTCAGTCAGACAAA	aaaaTTTGTCTGACTGACTGGAGG
2-5	acggAGAAACAAGAAGGCTCAGGA	aaaaTCCTGAGCCTTCTTGTTTCT
2-6	acggGTGTAATCCTATGAGCATTA	aaaaTAATGCTCATAGGATTACAC
2-7	acggGTGTGGGATGATGTATAGTG	aaaaCACTATACATCATCCCACA
2-8	acggACAGTTGGTTTCACTAAAGC	aaaaGCTTTAGTGAAACCAACTGT
2-9	acggGTTTCACTAAAGCAACTCAA	aaaaTTGAGTTGCTTTAGTGAAAC
2-10	acggCTTTAGTGAAACCAACTGTT	aaaaAACAGTTGGTTTCACTAAAG
X-3-1	acggATGGGGGAAAAATTGTGGTA	aaaaTACCACAATTTTTCCCCCAT
X-3-2	acggCAAAGATGGACATGTTTAAA	aaaaTTTAAACATGTCCATCTTTG
X-3-3	acggCTAGAGTAATGGCCAGTGTA	aaaaTACACTGGCCATTACTCTAG
X-3-4	acggCATTACAGGTTTATTTCCTC	aaaaGAGGAAATAAACCTGTAATG
X-3-5	acggTGTGACTTCCATTACAGGTT	aaaaAACCTGTAATGGAAGTCACA
C-3-1	acggAGGCACATGAGATAATATGA	aaaaTCATATTATCTCATGTGCCT
C-3-2	acggCATTAATCTCACATCTCATC	aaaaGATGAGATGTGAGATTAATG
C-3-3	acggGCTTCACAAATAGACAAGTC	aaaaGACTTGTCTATTTGTGAAGC
C-3-4	acggCATAAGTTCAACTGACACAA	aaaaTTGTGTCAGTTGAACTTATG
C-3-5	acggCCAAAAGGTTGTTCACGTGA	aaaaTCACGTGAACAACCTTTTGG

sgRNA名称 sgRNA name	sgRNA成功出现缺失 sgRNA successfully deletion	缺失效率/% Deletion efficiency
1-3	4/10	40
1-9	9/15	60
2-2	5/15	34
2-6	9/11	84
3-1	4/16	25
3-2	2/7	28

华西牛XIST敲除 XIST knockout in Huaxi cattle	96孔板数量 Number of 96-well plates	48孔板数量 Number of 48-well plates	24孔板数量 Number of 24-well plates	筛选单克隆数 Monoclonal numbers	验证成功 Successful authentication
284方案一284 option one	15	16	16	320	5
284方案二284 option two	15	7	6	165	1
284方案三284 option three	15	6	6	73	0

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