利用CRISPR/Cas9技术制备BLG基因敲除牛乳腺上皮细胞系

doi:10.11843/j.issn.0366-6964.2024.10.020

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (10): 4475-4488.doi: 10.11843/j.issn.0366-6964.2024.10.020

利用CRISPR/Cas9技术制备BLG基因敲除牛乳腺上皮细胞系

丁修虎¹^,²(), 林志平³, 赵芳¹, 陈坤琳¹, 仲跻峰¹, 张燕⁴, 高运东⁴, 李惠侠², 王慧利¹, 张建丽¹^,*(), 丁强¹^,*()

1. 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
2. 南京农业大学动物科技学院, 南京 210095
3. 江苏优源奶业产业研究院有限公司, 南京 211100
4. 山东奥克斯畜牧种业有限公司, 济南 250100

收稿日期:2024-04-01 出版日期:2024-10-23 发布日期:2024-11-04
通讯作者: 张建丽,丁强 E-mail:dxh1825047521@163@.com;zhangjianli79@163.com;dingqiang198907@163.com
作者简介:丁修虎(1998-), 男, 安徽滁州人, 硕士, 主要从事牛的分子育种研究, E-mail:dxh1825047521@163@.com
基金资助:
江苏省重点研发项目“基于新型单碱基编辑器的奶牛高效精准育种技术研究”(BE2021372);江苏省农业科学院探索性颠覆性创新计划项目(ZX(21)1214)

Highly Efficient BLG Knockout in Bovine Mammary Epithelial Cells by Using CRISPR/Cas9

Xiuhu DING¹^,²(), Zhiping LIN³, Fang ZHAO¹, Kunlin CHEN¹, Jifeng ZHONG¹, Yan ZHANG⁴, Yundong GAO⁴, Huixia LI², Huili WANG¹, Jianli ZHANG¹^,*(), Qiang DING¹^,*()

1. 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
2. College of Animal Science & Technology, Nanjing Agricultural University, Nanjing 210095, China
3. Jiangsu Youyuan Dairy Research Institute Co., Ltd., Nanjing 211100, China
4. Shandong OX Livestock Breeding Co., Ltd., Jinan 250100, China

Received:2024-04-01 Online:2024-10-23 Published:2024-11-04
Contact: Jianli ZHANG, Qiang DING E-mail:dxh1825047521@163@.com;zhangjianli79@163.com;dingqiang198907@163.com

摘要/Abstract

摘要：

旨在利用CRISPR/Cas9系统构建BLG基因敲除牛乳腺上皮细胞系(bovine mammary epithelial cells, bMECs)，同时检测BLG基因敲除后对细胞的影响。本研究利用体外切割试验筛选获得靶向牛的BLG基因的第一外显子序列的3条sgRNA，与CRISPR/Cas9表达载体电转染入bMECs，利用浓度为1.8 μg·mL^-1嘌呤霉素和6 μg·mL^-1稻瘟菌素双药筛和PCR分析鉴定获得BLG基因编辑的单克隆细胞株；利用Western blot(WB)验证细胞BLG表达；绘制生长曲线和CCK-8试验检测BLG基因敲除对bMECs细胞增殖的影响；同时根据sgRNA序列，对基因组上潜在脱靶位点进行PCR测序分析。经筛选和PCR序列分析，共筛选获得了9株BLG基因编辑细胞株，其编辑效率为90%(9/10)，其中4株细胞株BLG基因大片段删除。序列分析结果显示，单克隆细胞株编辑位点呈现片段插入缺失和碱基替换等多种编辑类型，WB结果显示敲除细胞株BLG蛋白表达显著降低，且细胞生长曲线和CCK-8检测结果表明BLG基因敲除的细胞生长速度加快。本研究利用CRISPR/Cas9技术成果构建了牛乳腺上皮细胞BLG基因高效敲除方法，BLG基因敲除后可显著影响牛乳腺上皮细胞的增殖，为探究BLG敲除牛在乳腺上皮细胞水平探究其功能机制提供细胞模型。

关键词: BLG基因, 牛乳腺上皮细胞系, CRISPR/Cas9, 基因编辑

Abstract:

This study aimed to establish CRISPR/Cas9 mediated BLG-knockout (BLG-KO) system in bovine mammary epithelial cells (bMECs), and to explore the function effect of BLG-KO on bMECs. Three sgRNAs were designed from the first exon of the bovine BLG gene sequence and screened by using in vitro cleavage test. The Cas9 expression vector and sgRNAs vectors were cotransfected into bMECs by electroporation. Monoclonal cell lines were screened with 1.8 μg·mL^-1 puromycin and 6 μg·mL^-1 blasticidin. BLG-KO gene editing type were verified by using PCR amplification and sequencing analysis. The potential off-target sites were selected according to the sgRNA sequence and detected by PCR sequencing. The BLG expressing level of knockout cell was detected by Western blot (WB). Cell viability was tested by cell counting kit-8 (CCK-8) assay, further the growth curve was drawn to analyze the proliferation effect of bMECs cells after BLG-KO. Two sgRNAs were screened and used for further gene edit. 10 monoclonal cell lines were obtained after screening and 9 BLG-edit lines verified by PCR sequencing, 4 clones with large fragment deleted at BLG sequence. Sequence analysis showed that the editing sites contained multiple gene-editing types, including insertion, deletion and base substitution. WB result showed that BLG-KO cell lines with low BLG protein expressing level compare to none edit cell. Furthermore, the BLG-KO promote cell growth and proliferation according to the results of cell growth curve and CCK-8 assay. In this study, we constructed an efficient BLG-KO method in bMECs by using CRISPR/Cas9 system, and the results demonstrated that BLG-KO significantly affected the cell proliferation in bMECs, the results provided a cellular model for exploring the functional mechanism for BLG-KO cattle.

Key words: BLG gene, bMECs, CRISPR/Cas9, gene editing

中图分类号:

S823.2

丁修虎, 林志平, 赵芳, 陈坤琳, 仲跻峰, 张燕, 高运东, 李惠侠, 王慧利, 张建丽, 丁强. 利用CRISPR/Cas9技术制备BLG基因敲除牛乳腺上皮细胞系[J]. 畜牧兽医学报, 2024, 55(10): 4475-4488.

Xiuhu DING, Zhiping LIN, Fang ZHAO, Kunlin CHEN, Jifeng ZHONG, Yan ZHANG, Yundong GAO, Huixia LI, Huili WANG, Jianli ZHANG, Qiang DING. Highly Efficient BLG Knockout in Bovine Mammary Epithelial Cells by Using CRISPR/Cas9[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4475-4488.

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sgRNA序列(5′→3′)(斜体为PAM区) sgRNA sequence (Italic indicate PAM)		引物序列(5′→3′) sgRNA primers sequence
sgRNA1(sg1)	CACCCAGACCATGAAGGGCCTGG	F: ACCGCACCCAGACCATGAAGGGCC R: AAACGGCCCTTCATGGTCTGGGTG
sgRNA2(sg2)	GCACTTCATGGCTGCAGCTGGGG	F: ACCGGCACTTCATGGCTGCAGCTG R: AAACCAGCTGCAGCCATGAAGTGC
sgRNA3(sg3)	TGGATATCCAGAAGGTTCGAGGG	F: ACCGTGGATATCCAGAAGGTTCGA R: AAACTCGAACCTTCTGGATATCCA

名称 Name	引物序列(5′→3′) Primers sequence
IVT-sg1-F	$\underline{{\rm{TAATACGACTCACACTATA}}}$CACCCAGACCATGAAGGGCC
IVT-sg1-R	AAAAGCACCGACTCGGTGCC
IVT-sg2-F	$\underline{{\rm{TAATACGACTCACACTATA}}}$GGCACTTCATGGCTGCAGCTG
IVT-sg2-R	AAAAGCACCGACTCGGTGCC
IVT-sg3-F	$\underline{{\rm{TAATACGACTCACACTATA}}}$GTGGATATCCAGAAGGTTCGA
IVT-sg3-R	AAAAGCACCGACTCGGTGCC

分组 Group	1	2	3	4	5	6	7	8	9	10	11	12
嘌呤霉素浓度/(μg·mL^-1) Puromycin concentration	0	0.4	0.8	1.2	1.4	1.6	1.8	2.0	2.2	2.4	2.6	3.0
稻瘟菌素浓度/(μg·mL^-1) Blasticidin concentration	0	1	2	3	4	5	6	7	8	9	10	11

潜在脱靶位点 Potential off-target site	潜在脱靶位点序列(5′→3′) Potential off-target site sequence	方向 Direction	错配碱基 Mismatched bases	染色体 Chromosome
sgRNA1	CACCCAGACCATGAAGGGCCTGG
sg1-OT-1	gACCCAGACCATtAAGGGCCGGG	+	2MMS (1:13)	chr4
sg1-OT-2	gACCCAGtCCATGAAGGGCCTGG	+	2MMS (1:8)	chr14
sg1-OT-3	CtCCCAGACCAgGAAGGtCCAGG	+	3MMS (2:12:18)	chr17
sg1-OT-4	CACCCAaACCAaGAAGaGCCAGG	+	3MMS (7:12:17)	chr3
sg1-OT-5	gAgCCAGACCATaAAGGGCCTGG	+	3MMS (1:3:13)	chr2
sgRNA3	TGGATATCCAGAAGGTTCGAGGG
sg3-OT1	TGGATATCCAGgAGGTTaGAGGG	+	2MMS (12:18)	chr18
sg3-OT2	gGGAaATCCAGAAGGTTtGAAGG	+	3MMS (1:5:17)	chr8
sg3-OT3	TtGATATCCAGAAGGTTgtAAGG	+	3MMS (2:18:19)	chr15
sg3-OT4	TGGAcATCCtGAAGGTTaGAAGG	-	3MMS (5:10:18)	chr5
sg3-OT5	TGGATAcCCAGtAGGgTCGAAGG	+	3MMS (7:12:16)	chr24

靶位点 Target site	引物序列(5′→3′) Primers sequence		产物大小/bp Product size
靶位点 Target site	上游引物 Forward	下游引物 Reverse	产物大小/bp Product size
sg1-OT-1	GTGCAAATCTTCATCCATGCTG	AGGCCTCTGTTTCTCATCACA	511
sg1-OT-2	AGCGTCGCAGAAATCAGAAC	TCCCTAGTGGCTAAGATGGTAAA	410
sg1-OT-3	ACTCTTACAGGTGGTTGGTTTGC	TGGTCCTGCCATGCTGATT	418
sg1-OT-4	TGGCTGGGTATCACATGCAG	GGGAGACGTCAATCGTGGTT	481
sg1-OT-5	GCCAGGCCTTACACACTACA	TCCCCTCCTGGTTTGAGTCT	349
sg3-OT-1	TGCTCAGCCCCACTATCTCC	CCATAGACGGCAGCCCAAT	334
sg3-OT-2	ACCCATTTGTTCATCCATCTCC	TGTCCTCCCCTTCCATCTTT	416
sg3-OT-3	AATGGCAAGCCCCTCCT	AGCCCATTCTTTCCTCAGTTT	410
sg3-OT-4	GACTTCAGGCCCTGTCCTTG	TGCCTGTAAGATGAGTGTGGG	504
sg3-OT-5	AGGCAGGGTTTTAGGTTTCC	ATTCTCCTCCTTAACATGGGTG	373

利用CRISPR/Cas9技术制备BLG基因敲除牛乳腺上皮细胞系

Highly Efficient BLG Knockout in Bovine Mammary Epithelial Cells by Using CRISPR/Cas9

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参考文献 40

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细胞总数 Total cell count	荧光细胞数 Number of fluorescent cells	转染效率/% Transfection efficiency
431	179	41.5

编号 Number	sgRNA1	sgRNA3	编辑类型 Type	移码 Frameshift
WT	CACCCAGACCATGAAGGGCC	TGGATATCCAGAAGGTTCGA
1	CACCCAGACCATGAAGGGGCC	gGaAaATCCAGAAGGTTTCGA	+2 bp	3n+2
2	CACCCAGACCgTGAA------	------TGCACAGGGT------	-19 bp	3n+2
3	CACCCAGACCATGAAGGGGCC	gGaATATCCAGAAGGTTTCGA	+2 bp	3n+2
4&8	--------------------	--------------------	大片段缺失
6	CACCCAGACCATGAAGGGGCC	cGGTATCCCAgAAGGTTTCaA	+3 bp	3n
7	CACCCAGACCATGAAGGGCC	TGGATATCCAGgAccggCGA		3n
9	CACCCAGACCATGAAGGGCC	TGGATATTCCgGAgGGTGTCGA	+2 bp	3n+2
10	CACCCAGACCATGAA------	---------------------	大片段缺失

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编号 Number	sgRNA1	sgRNA3	插入编辑类型 Indes forms	移码 Frameshift	占比 Proportion
WT	CACCCAGACCATGAAGGGCC	TGGATATCCAGAAGGTTCGA	wild type
2-1	CACCCAGACCATG-----	CCTGGATATCCAGAAaa--	-5 bp	3n+2	1/12
2-2	CACCCAGACCATGAAGGGCC	CCTGGATATCCAGAAGGTT			5/12
2-3	CACCCAG-------------CC	TGGATATCCAGAAGGT----	-17 bp	3n+2	2/12
2-4	CACCCAGACCATG-----CC	TGGATATCCAGAAG--TCGA	-7 bp	3n+2	1/12
2-5	CACCCAGACCATGAAGGGCC	TGGATATCC-----------	-11 bp	3n+2	1/12
2-6	CACCCAGACCATGAAGGGGCC	TGGATATCCAGAAGGTTTCGA	+2 bp	3n+2	1/12
2-7	CgCCCAG-----------CC	TGGATATCCAGAAGGT----	-15 bp	3n	1/12
8-1	------------------------	------------------------	大片段缺失		12/12
10-1	CACCCAGACCATGAAG--CC	TGGATATCCAGAAGGTTTCG	-1 bp	3n+1	4/12
10-2	CACCCAGACCATGAAGGGCC	TGGATATCCAGAAGGTTCGA			1/12
10-3	CACCCAGACCATG----GCC	TGGATATCCAGAAGGTTTCGA	-3 bp	3n	3/12
10-4	CACCCAGACCATG----GCC	TGGATATCCAGAAGGGTTCGA	-3 bp	3n	1/12