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

Abstract

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

CLC Number:

S823.2

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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References 40

1	HOCHWALLNERH,SCHULMEISTERU,SWOBODAI,et al.Cow's milk allergy: from allergens to new forms of diagnosis, therapy and prevention[J].Methods,2014,66(1):22-33. doi: 10.1016/j.ymeth.2013.08.005
2	LEMOSL,ASSISH C,ALVESJ L,et al.Neuroimmune circuits involved in β-lactoglobulin-induced food allergy[J].Brain Behav Immun Health,2022,23,100471. doi: 10.1016/j.bbih.2022.100471
3	SCHOEMAKERA A,SPRIKKELMANA B,GRIMSHAWK E,et al.Incidence and natural history of challenge-proven cow's milk allergy in European children—EuroPrevall birth cohort[J].Allergy,2015,70(8):963-972. doi: 10.1111/all.12630
4	GAIN,UNIACKE-LOWET,O'REGANJ,et al.Effect of protein genotypes on physicochemical properties and protein functionality of bovine milk: a review[J].Foods,2021,10(10):2409. doi: 10.3390/foods10102409
5	GIANNETTIA,TOSCHI VESPASIANIG,RICCIG,et al.Cow's milk protein allergy as a model of food allergies[J].Nutrients,2021,13(5):1525. doi: 10.3390/nu13051525
6	ABD EL-SALAMM H,EL-SHIBINYS.Preparation, properties, and uses of enzymatic milk protein hydrolysates[J].Crit Rev Food Sci Nutr,2017,57(6):1119-1132. doi: 10.1080/10408398.2014.899200
7	王明礼,钱珊珊,李艾黎,等.降低牛乳致敏性方法的研究进展[J].中国乳品工业,2021,49(7):25-31.
	WANGM L,QIANS S,LIA L,et al.Research progress on methods of reducing allergenicity of milk[J].China Dairy Industry,2021,49(7):25-31.
8	吴雯倩,左姣丽,肖冰,等.热处理对蛋白质的影响[J].食品安全导刊,2015,(36):45.
	WUW Q,ZUOJ L,XIAOB,et al.Effect of heat treatment on proteins[J].China Food Safety Magazine,2015,(36):45.
9	HATTORIM,MIYAKAWAS,OHAMAY,et al.Reduced immunogenicity of β-lactoglobulin by conjugation with acidic oligosaccharides[J].J Agric Food Chem,2004,52(14):4546-4553. doi: 10.1021/jf0353887
10	LIUY,COTTLEW T,HAT.Mapping cellular responses to DNA double-strand breaks using CRISPR technologies[J].Trends Genet,2023,39(7):560-574. doi: 10.1016/j.tig.2023.02.015
11	BUW,CREIGHTONC J,HEAVENERK S,et al.Efficient cancer modeling through CRISPR-Cas9/HDR-based somatic precision gene editing in mice[J].Sci Adv,2023,9(19):eade0059. doi: 10.1126/sciadv.ade0059
12	WANGY,QIT,LIUJ T,et al.A highly specific CRISPR-Cas12j nuclease enables allele-specific genome editing[J].Sci Adv,2023,9(6):eabo6405. doi: 10.1126/sciadv.abo6405
13	CONGL,RANF A,COXD,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013,339(6121):819-823. doi: 10.1126/science.1231143
14	ZHANGX M,LIW R,WUY S,et al.Disruption of the sheep BMPR-IB gene by CRISPR/Cas9 in in vitro-produced embryos[J].Theriogenology,2017,91,163-172. doi: 10.1016/j.theriogenology.2016.10.025
15	WANGX L,NIUY Y,ZHOUJ K,et al.Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep[J].Sci Rep,2016,6,32271. doi: 10.1038/srep32271
16	CRISPOM,MULETA P,TESSONL,et al.Efficient generation of Myostatin knock-out sheep using CRISPR/Cas9 technology and microinjection into zygotes[J].PLoS One,2015,10(8):e0136690. doi: 10.1371/journal.pone.0136690
17	ZHOUS W,KALDSP,LUOQ,et al.Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality[J].BMC Genomics,2022,23(1):348. doi: 10.1186/s12864-022-08594-6
18	LIW R,LIUC X,ZHANGX M,et al.CRISPR/Cas9-mediated loss of FGF5 function increases wool staple length in sheep[J].FEBS J,2017,284(17):2764-2773. doi: 10.1111/febs.14144
19	ZHOUW J,WANY J,GUOR H,et al.Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9[J].PLoS One,2017,12(10):e0186056. doi: 10.1371/journal.pone.0186056
20	李炜杰,杨娇,王聪慧,等.电转液L对绵羊成纤维细胞电转染条件优化[J].新疆农业科学,2015,52(8):1481-1485.
	LIW J,YANGJ,WANGC H,et al.Optimization of an electrotransfer solution-L for Ovis Aries fibroblasts[J].Xinjiang Agricultural Sciences,2015,52(8):1481-1485.
21	董海龙,吴琼,刘明超,等.奶牛乳腺上皮细胞的体外分离培养及超微结构鉴定[J].中国兽医学报,2016,36(10):1763-1768.
	DONGH L,WUQ,LIUM C,et al.Inprimary culture and ultrastructural changes of bovine mammary epithelial cells[J].Chinese Journal of Veterinary Science,2016,36(10):1763-1768.
22	BAES,PARKJ,KIMJ S.Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases[J].Bioinformatics,2014,30(10):1473-1475. doi: 10.1093/bioinformatics/btu048
23	YUS L,LUOJ J,SONGZ Y,et al.Highly efficient modification of beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle[J].Cell Res,2011,21(11):1638-1640. doi: 10.1038/cr.2011.153
24	崔趁趁. TALEN介导的β-乳球蛋白基因敲除奶山羊的生产[D]. 杨凌: 西北农林科技大学, 2015.
	CUI C C. TALEN-mediated gene targeting of the β-lactoglobulin gene in goat[D]. Yangling: Northwest A&F University, 2015. (in Chinese)
25	周文君. 利用CRISPR/Cas9技术敲除山羊BLG基因与定点整合hLF基因的研究[D]. 南京: 南京农业大学, 2017.
	ZHOU W J. Knocking out goat BLG gene and intergration of hLF site-specifically using CRISP/Cas9 technology[D]. Nanjing: Nanjing Agricultural University, 2017. (in Chinese)
26	张曼. 奶山羊BLG基因调控序列指导的乳腺特异性表达载体的构建和优化[D]. 杨凌: 西北农林科技大学, 2015.
	ZHANG M. Construction and optimizing of gland-specific expression vector by dairy goat BLG gene regulatory elements[D]. Yangling: Northwest A&F University, 2015. (in Chinese)
27	丁修虎, 丁强, 王悦, 等. 利用胞嘧啶碱基编辑器高效制备奶牛BLG基因敲除胚胎的研究[J/OL]. 南京农业大学学报, 1-10[2024-04-10]. http://kns.cnki.net/kcms/detail/32.1148.S.20240507.1850.008.html.
	DING X H, DING Q, WANG Y, et al. Highly efficient BLG gene knock out using base editors in cattle embryos[J/OL]. Journal of Nanjing Agricultural University, 1-10[2024-04-10]. http://kns.cnki.net/kcms/detail/32.1148.S.2240507.1850.008.html. (in Chinese)
28	张家翔,韩佃刚,师亚玲,等.利用CRISPR/Cas9技术构建PPARγ基因敲除的IPEC-J2细胞系[J].中国畜牧兽医,2023,50(7):2670-2677.
	ZHANGJ X,HANGD G,SHIY L,et al.Construction of IPEC-J2 cell lines with PPARγ gene knockout mediated by CRISPR/Cas9 technology[J].China Animal Husbandry & Veterinary Medicine,2023,50(7):2670-2677.
29	LIJ,KONGD L,KEY P,et al.Application of multiple sgRNAs boosts efficiency of CRISPR/Cas9-mediated gene targeting in Arabidopsis[J].BMC Biol,2024,22(1):6. doi: 10.1186/s12915-024-01810-7
30	杨花,刘孜斐,吕文莉,等.基于CRISPR/Cas9系统构建绵羊VASA基因敲入载体及验证[J].生物工程学报,2023,39(10):4219-4233.
	YANGH,LIUZ F,LÜW L,et al.Construction and validation of sheep VASA gene knock-in vector based on CRISPR/Cas9 system[J].Chinese Journal of Biotechnology,2023,39(10):4219-4233.
31	ZHANGY R,YINT L,ZHOUL Q.CRISPR/Cas9 technology: applications in oocytes and early embryos[J].J Transl Med,2023,21(1):746. doi: 10.1186/s12967-023-04610-9
32	张晨俭,李隐侠,丁强,等.CRISPR/Cas9技术高效制备山羊SOCS2基因编辑胚胎[J].畜牧兽医学报,2024,55(1):129-141. doi: 10.11843/j.issn.0366-6964.2024.01.014
	ZHANGC J,LIY X,DINGQ,et al.Efficient preparation of CRISPR/Cas9-mediated goat SOCS2 gene edited embryos[J].Acta Veterinaria et Zootechnica Sinica,2024,55(1):129-141. doi: 10.11843/j.issn.0366-6964.2024.01.014
33	ZHANGX H,TEEL Y,WANGX G,et al.Off-target effects in CRISPR/Cas9-mediated genome engineering[J].Mol Ther Nucleic Acids,2015,4(11):e264.
34	CHENX Y,XUF,ZHUC M,et al.Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans[J].Sci Rep,2014,4(1):7581. doi: 10.1038/srep07581
35	DOENCHJ G,FUSIN,SULLENDERM,et al.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9[J].Nat Biotechnol,2016,34(2):184-191. doi: 10.1038/nbt.3437
36	SMITHC,GOREA,YANW,et al.Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs[J].Cell Stem Cell,2014,15(1):12-13. doi: 10.1016/j.stem.2014.06.011
37	MAS Y,WANGA M,CHENX X,et al.Deep sequencing reveals the comprehensive CRISPR-Cas9 editing spectrum in Bombyx mori[J].CRISPR J,2021,4(3):371-380.
38	WANGD Q,ZHANGC D,WANGB,et al.Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning[J].Nat Commun,2019,10(1):4284.
39	HENDELA,BAKR O,CLARKJ T,et al.Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells[J].Nat Biotechnol,2015,33(9):985-989.
40	TAIC S,CHENY Y,CHENW L.β-Lactoglobulin influences human immunity and promotes cell proliferation[J].Biomed Res Int,2016,2016,7123587.

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

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

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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