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

Abstract

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

CLC Number:

S823.2

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

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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