CRISPR/Cas9体外酶切检测猪生长抑素基因定点修饰靶点活性的研究

doi:10.16431/j.cnki.1671-7236.2016.01.005

Abstract

Abstract: This study was aimed to detect the targets activities which were modified in pig SST gene through the CRISPR/Cas9 enzyme digestion method in vitro.Five 20 bp sgRNAs SST-sgRNA-g1, SST-sgRNA-g2, SST-sgRNA-g3, SST-sgRNA-g4 and SST-sgRNA-g5 of SST were designed.Oligonucleotide sequences of sgRNA were chemically synthesized, and then inserted into linearized plasmid which could express Cas9 and sgRNA together.Then, the correct sgRNA plasmids were selected as a template for transcription of SST-sgRNA in vitro.Target DNA fragments were digested by CRISPR/Gas9in vitro and then the gray bands of enzyme digestion were converted into sgRNA activity.The results indicated that the target nucleotide sequences were successfully inserted into the expected sites of vector and sequences were correct.The vector was successfully transcribed into sgRNA in vitro.It showed that compared with standard sgRNA1 and sgRNA2 enzyme activity, the target SST-sgRNA-g1, SST-sgRNA-g4 and SST-sgRNA-g5 had high activities, they could provide basis for the site-directed modification of gene in cell and embryo.

Key words: pig; SST gene; Cas9 enzyme; single-guide RNA(sgRNA); transcription in vitro

CLC Number:

LI Xiao-min, REN Hong-yan, BI Yan-zhen, LIU Xi-mei, ZHENG Xin-min, WU Min-yao. Study on Detection of Pig SST Gene Site-directed Modification Activity by CRISPR/Cas9 System in vitro[J]. , 2016, 43(1): 31-38.

References

[1] 江青东,王艳玲,王月影.生长抑素及其受体的研究进展[J].动物医学进展,2005,26(6):25-28.
[2] 江青东.生长抑素在畜牧生产中的应用研究进展[J].上海畜牧兽医通讯,2007,3:10.
[3] 范志勇,王康宁,周定刚.生长抑素的作用及调控技术研究进展[J].中国饲料,2003,19:5-7.
[4] 孙科,邹晓庭.生长抑素及在畜牧生产上的应用[J].江西饲料,2008,2:1-3.
[5] 赵学军,苏军,刘建新.生长抑素在动物生产上的研究进展[J].饲料博览,2002,9:6-9.
[6] 官琼,朱崇淼,季伟.生长抑素调控技术在畜牧业中的研究及应用[J].福建畜牧兽医,2005,27(2):19-21.
[7] 崔文涛,单同领,李奎.转基因猪的研究现状及应用前景[J].中国畜牧兽医,2007,34(4):58-62.
[8] Sorek R,Kunin V,Hugenholtz P.CRISPR——A widespread system that provides acquired resistance against phages in bacteria and archaea[J]. Microbiology,2008,3(6):181-186.
[9] 左其生,李东,张亚妮,等.CRISPR-CAS介导的基因编辑工具[J].生物技术通报,2014,7:37-43.
[10] Wiedenheft B,sternberg S H,Doudna J A.RNA-guided genetic silencing systems in bacteria and archaea[J]. Nature,2012,482(7385):331-338.
[11] 贾良杰.CRISPR/CAS基因组靶向编辑技术综述[J].中国医学导报,2014,11(22):154-156.
[12] 刘思也,夏海滨.一种新的由CRISPR/CAS介导的基因组靶向修饰技术[J].中国生物工程杂志,2013,33(10):117-123.
[13] Makarova K S,Critshin N V,Shabalina S A,et al.A putative RNA-interference-based immune system in prokaryotes:Computational analysis of the predicted enzymatic machinery,functional analogies with eukaryotic RNAi,and hypothetical mechanisms of action[J]. Biology Direct,2006,1(1):7.
[14] Cong L,Ran F A,Cox D,et al.Multiple genome engineering using CRISPR/Cas systems[J]. Science, 2013,339(6121):819-823.
[15] Mali P,Yang L,Esvelt K M,et al.RNA-guide human genome engineering via Cas9[J]. Science,2013,339(6121):823-826.
[16] Qi L S,Larson M H,Gilbert L A,et al.Repurposing CRISPR as an RNA-guided platform for seguence-specific control of gene expression[J]. Cell,2013,152(5):1173-1183.
[17] Bassett A R,Liu J L.CRISPR/Cas9 and genome editing in Drosophila[J]. Journal of Genetics and Genomics,2014,41(1):7-19.
[18] 梁振伟,饶书权,沈岩,等.通过CRISPR/Cas9系统敲除人源PDE 10A基因[J].基础医学与临床,2014,34(4):439-443.

Study on Detection of Pig SST Gene Site-directed Modification Activity by CRISPR/Cas9 System in vitro

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	WEI Mingbang, BIANBA Qiongda, XIAO Qingqing, DUAN Mengqi, CHAMBA Yangzom, SHANG Peng. Cloning,Bioinformatics and Expression Analysis of CDKN1B Gene in Tibetan Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2196-2206.
[2]	ZHANG Fangwei, ZHANG Qi, LEI Liangliang, SUN Wusheng, ZHANG Di, ZHANG Yunpeng, ZHANG Jingbo, WANG Xiuquan, ZHANG Jing, ZHANG Shumin. Polymorphism of HBEGF Gene and Its Association with Reproductive Traits in Songliao Black Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2370-2379.
[3]	PAN Pengcheng, LEI Zongquan, LI Xian, HU Xiangyun, QIN Qiantao, QIN Zhaoxian, GUAN Zhihui, CHEN Baojian, XIE Bingkun. Bioinformatics Analysis,Eukaryotic Expression Vector Construction and Tissue Expression of MYL2 Gene in Luchuan Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1796-1806.
[4]	OUYANG Kun, WEI Limin, SUN Ruiping, XIE Yali, QI Qi, LI Xiang, LIU Quanwei, CHAO Zhe, ZHANG Yongliang, CHEN Ting. Effects of Curcumin on Growth Performance,Feed Apparent Digestibility and Serum Biochemical Indexes in Hainan Tunchang Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1867-1875.
[5]	WU Jingxuan, SUN Zheng, MA Xuelian, ZHANG Liyang, LIAO Xiudong, LUO Xugang, LI Sufen, LYU Lin. A Survey on Magnesium Contents in Feed Resources for Livestock and Poultry in China [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1888-1898.
[6]	WANG Zeping, WANG Feifei, ZHANG Ruiming, FU Yanfeng, ZHAO Weimin, DAI Chaohui, CHENG Jinhua, LIAO Chao, LI Hui. Analysis on Polymorphism of SLA-1 Gene and Its Disease Resistance Potential in Suzi Pig [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1918-1927.
[7]	LIU Hongrun, ZHU Siran, FENG Lingli, ZHANG Kun, YAN Gang, WANG Yubin, ZHANG Shuai, JIANG Shan, XU Di, LAN Ganqiu, LIANG Jing. Cloning,Bioinformatics Analysis and Tissue Expression Localization of CDO1 Gene in Large White Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1352-1363.
[8]	YAO Kang, CAO Shuting, JIANG Zongyong, WANG Li. The Research Progress of Lysine Application and Requirement in Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1364-1374.
[9]	ZHANG Rui, MU Chunyu, TANG Qingping, BU Zhu, YANG Mingjun, XIA Aiping, YANG Xiaoming. Polymorphism of GnIH Gene and Its Correlation with Egg-laying Traits in Pigeons [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1412-1421.
[10]	ZHANG Fangwei, ZHANG Qi, ZHANG Yunpeng, LI Xin, ZHANG Jingbo, ZHANG Shumin, YU Yongsheng. Polymorphism of FOXA2 Gene Exon 3 and Its Association with Reproductive Traits in Songliao Black Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1444-1451.
[11]	TAN Yuancheng, LUO Hualun, ZHANG Yiyu, YANG Suan, WANG Yingtong, WANG Chunyuan. Polymorphism of MyoG Gene and Its Association with Body Size and Meat Traits in White Feather King Pigeons [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1461-1471.
[12]	HAN Dayong, ZHOU Chunbao, NI Ligang, CHEN Zhangyan, ZHAO Jinming. Genetic Diversity of mtDNA D-loop Sequence in Sujiang Pig [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1472-1479.
[13]	YANG Zhi, YU Jiaxin, YANG Linfu, WANG Kang, LI Guanzong, DUAN Gang, YANG Fumei, LIU Zhiqi, ZHOU Ke, HE Jun, DAI Feiyan. Study on the Therapeutic Effect of Modified Sini Decoction on Piglet Diarrhea [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1707-1717.
[14]	CHANG Yitong, ZHANG Wei, PENG Yinglin, CHEN Chen. Evolutionary Analysis,Target Gene Prediction and Tissue Expression Analysis of miR-192 [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 882-892.
[15]	LIU Zhen, ZHOU Xueqin, XU Fei, LI Zhefeng, SUN Hanxue, LI Guoqin, TAO Zhengrong, GUAN Mei, ZHENG Qiang, DIAO Xinping, LU Lizhi. Effects of Coated Sodium Butyrate on Growing Performance,Slaughter Performance, Immune Organ Index and Digestive Enzyme Activity of Breeding Pigeons and Squabs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 998-1006.