基于CRISPR/Cas9技术的牛乳腺上皮细胞全基因组敲除文库的构建

doi:10.11843/j.issn.0366-6964.2025.06.016

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (6): 2711-2723.doi: 10.11843/j.issn.0366-6964.2025.06.016

基于CRISPR/Cas9技术的牛乳腺上皮细胞全基因组敲除文库的构建

高林娜¹^,²(), 蒋影影²^,³, 王悦²^,³, 史倩倩¹^,², 安振江²^,³, 王慧利², 沈阳阳², 陈坤琳²^,*(), 张乐颖¹^,*()

1. 河北工程大学生命科学与食品工程学院, 邯郸 056038
2. 江苏省农业科学院畜牧研究所, 江苏省畜禽精准育种工程研究中心, 农业农村部种养结合重点实验室, 南京 210014
3. 南京农业大学动物科技学院, 南京 210095

收稿日期:2024-11-19 出版日期:2025-06-23 发布日期:2025-06-25
通讯作者: 陈坤琳,张乐颖 E-mail:13292751837@163.com;chenkunlin@jaas.ac.cn;zhangly056000@126.com
作者简介:高林娜(1997-)，女，河北邢台人，硕士，主要从事牛的分子育种研究，E-mail：13292751837@163.com
基金资助:
国家自然科学基金(32472872);江苏省重点研发项目(BE2021372)

Construction of a Whole Genome Knockout Library of bMECs Based on CRISPR/Cas9 Technology

GAO Linna¹^,²(), JIANG Yingying²^,³, WANG Yue²^,³, SHI Qianqian¹^,², AN Zhenjiang²^,³, WANG Huili², SHEN Yangyang², CHEN Kunlin²^,*(), ZHANG Leying¹^,*()

1. School of Life Sciences and Food Engineering, Hebei University of Engineering, Handan 056038, China
2. Key Laboratory of Crop and Animal Integrated Farming of Ministry of Agriculture and Rural Affairs, Jiangsu Province Engineering Research Center for Precision Animal Breeding, Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
3. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China

Received:2024-11-19 Online:2025-06-23 Published:2025-06-25
Contact: CHEN Kunlin, ZHANG Leying E-mail:13292751837@163.com;chenkunlin@jaas.ac.cn;zhangly056000@126.com

摘要/Abstract

摘要：

旨在构建奶牛乳腺CRISPR/Cas9全基因组敲除细胞文库，用于筛选与奶牛泌乳、应激及疾病等相关的功能基因。本研究靶向牛全基因组蛋白编码基因，利用CRISPR/Cas9技术设计合成转录sgRNAs文库，克隆至慢病毒载体LentiCRISPR-V2，建立牛全基因组CRISPR/Cas9敲除质粒文库，并进行gEditing ScreeningTM文库测序验证；然后将敲除质粒文库进行慢病毒包装和滴度测定，感染牛乳腺上皮细胞(bovine mammary epithelial cells，bMECs)后经嘌呤霉素抗性筛选及RT-qPCR、Western blot技术确定最佳感染效果，最终通过高通量测序验证敲除细胞库中sgRNA覆盖率确定细胞文库质量。本研究构建的全基因组敲除质粒文库共靶向20 545个蛋白编码基因，包含61 237条sgRNA和3 062条牛基因组上无靶点的sgRNA；gEditing ScreeningTM质粒文库测序结果显示，全基因组质粒文库覆盖率为100%，均一性为2.35，测序深度为577×；进一步对质粒文库进行慢病毒包装，获得滴度为7.01×10⁸ TU·mL^-1的病毒液并感染bMECs, 嘌呤霉素筛选14 d得到MOI=0.2、0.3、0.4、0.5的稳转细胞株，RT-qPCR、Western blot结果表明，MOI=0.2时感染效果最佳；高通量测序结果显示，细胞文库覆盖率为78.74%，碱基稳定、序列质量较高。综上所述，牛全基因组质粒文库、全基因组敲除细胞文库符合质量标准和试验需求，能为挖掘调控牛泌乳性状及乳腺健康的关键基因研究提供重要的细胞筛选平台。

关键词: 牛, CRISPR/Cas9, 全基因组, 敲除质粒文库, 牛乳腺上皮细胞, 敲除细胞文库

Abstract:

The objective was to construct a genome-wide CRISPR/Cas9 knockout library in bovine mammary epithelial cells, which would facilitate the identification of functional genes associated with milk production, stress responses, and disease susceptibility in cow. This study focused on the protein-coding genes of the cow whole genome. CRISPR/Cas9 technology was employed to design and synthesize a sgRNAs library, which was subsequently cloned into the LentiCRISPR-V2 lentiviral vector to construct a cow whole-genome CRISPR/Cas9 knockout plasmid library. The gEditing ScreeningTM library sequencing was performed for validation. Next, the knockout plasmid library was packaged into lentivirus particles, and the viral titer was determined. Following infection of bovine mammary epithelial cells (bMECs), the optimal infection efficiency was confirmed through puromycin resistance screening and validated using RT-qPCR and Western blot analyses. Finally, high-throughput sequencing was conducted to verify the quality of the cell library by assessing the coverage and distribution of sgRNAs in the knockout cell library. A total of 20 545 protein-coding genes were targeted in the whole-genome knockout plasmid library constructed in this study, including 61 237 sgRNAs, of which 3 062 were not previously annotated in the cow genome. Sequencing analysis of the gEditing ScreeningTM plasmid library revealed a 100% coverage rate, a homogeneity index of 2.35, and an average sequencing depth of 577×. The plasmid library was subsequently packaged into lentiviral vectors, yielding a viral stock with a titer of 7.01×10⁸ TU·mL^-1, which was used to infect bMECs. Following 14 days of puromycin selection, stable cell lines were established at multiplicities of infection (MOI) of 0.2, 0.3, 0.4, and 0.5, with an optimal MOI determined to be 0.2. High-throughput sequencing results indicated that the cell library achieved a coverage rate of 78.74%, with stable base composition and high-quality sequence reads. In conclusion, both the cow whole-genome plasmid library and the cow whole-genome knockout cell library have met the established quality standards and experimental requirements. These resources can serve as a critical platform for screening cells to investigate key genes that regulate cow lactation traits and mammary gland health.

Key words: cow, CRISPR/Cas9, whole genome, knockout plasmid library, bovine mammary epithelial cells, knockout cell library

中图分类号:

S823.2

高林娜, 蒋影影, 王悦, 史倩倩, 安振江, 王慧利, 沈阳阳, 陈坤琳, 张乐颖. 基于CRISPR/Cas9技术的牛乳腺上皮细胞全基因组敲除文库的构建[J]. 畜牧兽医学报, 2025, 56(6): 2711-2723.

GAO Linna, JIANG Yingying, WANG Yue, SHI Qianqian, AN Zhenjiang, WANG Huili, SHEN Yangyang, CHEN Kunlin, ZHANG Leying. Construction of a Whole Genome Knockout Library of bMECs Based on CRISPR/Cas9 Technology[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2711-2723.

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基因总数 Total number of genes	基因对应的sgRNAs数 Number of sgRNAs corresponding to genes	对照sgRNAs数 Number of control sgRNAs	sgRNAs总数 Total number of sgRNAs
20 545	61 237	3 062	64 299

引物名称Primer	引物序列(5′→3′)Sequence	片段长度/bp Length
β-actin	F：CATCGGCAATGAGCGGTTCC	144
β-actin	R：GTGTTGGCGTAGAGGTCCTTG	144
env	F：ATGATAGTAGGAGGCTTGGTAGGT	100
env	R：GGGTCTGAAACGATAATGGTGA	100
pol	F：ATGGCAGTATTCATCCACAATT	156
pol	R：GTCCCTGTAATAAACCCGAAA	156

引物名称Primer	引物序列(5′→3′)Sequence	片段长度/bp Length
sgRNA-pool	F: ATATCTTGTGGAAAGGACGAAACACCG	74
	R: TTAACTTGCTATTTCTAGCTCTAAAAC

样本名 Sample	sgRNA总数 Total number of sgRNAs	丢失sgRNA数目 Number of lost sgRNAs	sgRNA平均丰度 Average sgRNA abundance
质粒文库 Plasmid library	64 290	1	0.030 49

样品名称Sample	CT-均值CT-Mean	拷贝数Copy number
ALB-文库慢病毒液ALB-library lentivirus liquid	20.02	4.07×10⁴
RRE-文库慢病毒液RRE-library lentivirus liquid	24.61	4.65×10³
ALB-GFP参照病毒液ALB-GFP reference virus liquid	20.38	3.21×10⁴
RRE-GFP参照病毒液RRE-GFP reference virus liquid	30.32	1.31×10²

基于CRISPR/Cas9技术的牛乳腺上皮细胞全基因组敲除文库的构建

Construction of a Whole Genome Knockout Library of bMECs Based on CRISPR/Cas9 Technology

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样品名称 Sample	感染时细胞数目 Cell number	病毒使用量/mL Virus usage amount	病毒滴度/(TU·mL^-1) Virus titer
文库慢病毒Library lentivirus	1.60×10⁵	5.00×10^－5	7.30×10⁸
GFP参照病毒 GFP reference virus	1.60×10⁵	5.00×10^－5	2.61×10⁷
文库实际滴度 Actual titer of the library	/	/	7.01×10⁸

样本 Sample	测序长度/bp Length	sgRNA reads总数 Total sgRNA reads	实际sgRNA reads Mapped reads	覆盖率/% Coverage rate
细胞文库Cell library	150	32 029 040	25 218 095	78.74

基因名称Gene name	向导RNA(5′→3′)sgRNA	sgRNA的count数Count reads
GC	TTTGTTGGCTCTACGTAAGT	1 376 398
PALB2	TTTGTTGGCCGCCGGTCAGA	727 513
GUCY1A2	TTTGTTGGAATGGTCTGCAT	631 165
CELF3	TTTGTTGAGGAGAGCGCGGC	574 104
C1QTNF7	TTTGTTCTGGCTGGCGCTAG	451 616
POLR3H	TTTGTTCAACTCTTCGGCGA	398 934
ORMDL1	TTTGTTAAGGTCCAAGCAAC	372 299
LOC787250	TTTGTGTTTGAGGTCGAGCT	335 331
NC-sgRNA-1698	TTTGTGTGGGTAGGTCCGGG	281 296
LOC100336208	TTTGTGTCTGCTACTGTCAT	279 009
SCN2B	TTTGTGGTTCACGGTGTAGC	275 791
PRPF38A	TTTGTGGGTGGTGTCTACGG	275 601
EDAR	TTTGTGGGCGAGCTGTGGCG	272 757
CDHR1	TTTGTGGGCACGCCCTACTA	271 241
LOC112447450	TTTGTGGCTCTCATGGTTAT	252 815
DYSF_1	TTTGTGGATCCGCGTCCGCT	235 587
TAFA1_2	TTTGTGGATAAGTGCTTGCG	235 579
SNX4_3	TTTGTGGAAAGACGACGAAT	233 111
BRINP1_1	TTTGTGGAAAGACACCGTCA	227 912
FANCB_2	TTTGTGCGTACTCTCTTGAA	213 775

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