Construction and Activity Analysis of the PLIN1 Gene CRISPR/Cas9 Vector

doi:10.13560/j.cnki.biotech.bull.1985.2019-0357

Abstract

Abstract:

The objectives of this work are to design and validate the sgRNA being capable of targeted cleaving the PLIN1 gene and to lay a foundation for the establishment of a highly efficient PLIN1 gene-cleaved animal model. Three sgRNAs with favorable evaluation results were designed using predictive software. After adding T7 promoter,the corresponding sgRNA was transcribed in vitro to construct the digestive system and to verify the cleavage activity in vitro. The corresponding gene knockout plasmid vector was constructed,and the plasmid was transferred into 3T3-L1 preadipocytes by electroporation and induced to differentiate. The expression of PLIN1 mRNA in cells was detected by RT-PCR. The expression of PLIN1 protein in cells was detected by Western blot. Three sgRNAs of PLIN1 were successfully transcribed in vitro and the restriction enzyme system was constructed. The cleavage efficiency of sgRNA-PLIN1-1 was 61.8%±9.0%,and that of sgRNA-PLIN1-2 was 64.1%±9.6%. The cleavage efficiency of sgRNA-PLIN1-3 was 34.1%±7.2%,and that of sgRNA-PLIN1-3 group was significantly lower than that of sgRNA-PLIN1-1 group and sgRNA-PLIN1-2 group（P<0.05）. The plasmid vector of three sgRNAs was successfully constructed and electroporated into 3T3-L1 preadipocytes,and the transfection efficiency was about 30%. The expression of PLIN1 mRNA in each positive interference group（P<0.01）significantly decreased at the 4th day after differentiation. Compared with sgRNA-PLIN1-3 group,there was a significant difference in the expression level of PLIN1 mRNA in sgRNA-PLIN1-1 group and sgRNA-PLIN1-2 group. The expression of PLIN1 protein in each positive interference group significantly decreased（P<0.01）,and there was no significant difference in the expression of PLIN1 protein between the positive interference groups. In conclusion,all three sgRNAs may cleave the exon 2 of the PLIN1 gene in a target manner,and the cleavage activity of the gRNA-PLIN1-1 group and the sgRNA-PLIN1-2 group is slightly higher than that of the sgRNA-PLIN1-3 group. In vitro active cleavage experiments may well predict intracellular cleavage and are an effective means of screening highly active sgRNA.

Key words: PLIN1 gene, CRISPR/Cas9, sgRNA, vector construction, cleavage efficiency

XU Xiang, DONG Wei-peng, ZHANG Shao-hua, FENG Chen-yi, LIU Tian-fu, YAN Jiong. Construction and Activity Analysis of the PLIN1 Gene CRISPR/Cas9 Vector[J]. Biotechnology Bulletin, 2019, 35(11): 89-95.

References

[1] Hryhorowicz M, Lipiński, D, et al.CRISPR/Cas9 immune system as a tool for genome engineering[J]. Archivum Immunologiae et Therapiae Experimentalis, 2017, 65(3):233-240.
[2] Cong L, Ran FA, Cox D, et al.Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339:819-823.
[3] Zhang H, Mccarty N.CRISPR editing in biological and biomedical investigation[J]. Journal of Cellular Biochemistry, 2017, 118(12):4152-4162.
[4] Xu H, Xiao TF, Chen CH, et al.Sequence determinants of improved CRISPR sg RNA design[J]. Genome Res, 2015, 25:1147-1157.
[5] Tsai SQ, Zheng Z, Nguyen NT, et al.GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases[J]. Nat Biotechnol, 2015, 33:187-197.
[6] Farboud B, Meyer BJ.Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design[J]. Genetics, 2015, 199(4):959-971.
[7] Brasaemle DL.Thematic review series:adipocyte biology. The perilipin family of structural lipid droplet proteins:stabilization of lipid droplets and control of lipolysis[J]. J Lipid Res, 2007, 48(12):2547-2559.
[8] Brasaemle DL, Wolins NE.Packaging of fat:an evolving model of lipid droplet assembly and expansion[J]. J Biol Chem, 2012, 287(4):2273-2279.
[9] Sztalryd C, Kimmel AR.Perilipins:lipid droplet coat proteins adapted for tissue specific energy storage and utilization, and lipid cytoprotection[J]. Biochimie, 2014, 96:96-101.
[10] Zou L, Wang W, Liu S, et al.Spontaneous hypertension occurs with adipose tissue dysfunction in perilipin-1 null mice[J]. Biochim Biophys Acta, 2016, 1862(2):182-191.
[11] Nishimasu H, et al.Crystal structure of Cas9 in complex with guide RNA and target DNA[J]. Cell, 2014, 156(5):935-949.
[12] 王威仪, 张彬, 季汉华, 等. 脂滴与心力衰竭的研究进展[J]. 生理科学进展, 2018(1). 69-73.
[13] Zetsche B, Gootenberg JS, Abudayyeh OO, et al.Cpf1 is a single RNA-Guided endonuclease of a class 2 CRISPR-Cas system.[J]. Cell, 2015, 163(3):759-771.
[14] 吴曦, 周舒雅, 刘甦苏等. 高效切割Hipp11敲入位点的sgRNA设计及活性验证[J]. 实验动物科学, 2015, 32(5):26-30.

[1]	CHEN Xiao-ling, LIAO Dong-qing, HUANG Shang-fei, CHEN Ying, LU Zhi-long, CHEN Dong. Advances in CRISPR/Cas9 System Modifying Saccharomycescerevisiae [J]. Biotechnology Bulletin, 2023, 39(8): 148-158.
[2]	YANG Yu-mei, ZHANG Kun-xiao. Establishing a Stable Cell Line with Site-specific Integration of ERK Kinase Phase-separated Fluorescent Probe Using CRISPR/Cas9 Technology [J]. Biotechnology Bulletin, 2023, 39(8): 159-164.
[3]	SHI Wei-tao, YAO Chun-peng, WEI Wen-Kang, WANG Lei, FANG Yuan-jie, TONG Yu-jie, MA Xiao-jiao, JIANG Wen, ZHANG Xiao-ai, SHAO Wei. Establishment of MDH2 Knockout Cell Line Using CRISPR/Cas9 Technology and Study of Anti-deoxynivalenol Effect [J]. Biotechnology Bulletin, 2023, 39(7): 307-315.
[4]	LIU Xiao-yan, ZHU Zhen-liang, SHI Guang-yu, HUA Zi-yu, YANG Chen, ZHANG Yong, LIU Jun. Strategies to Optimize the Expression of Mammary Gland Bioreactor [J]. Biotechnology Bulletin, 2023, 39(5): 77-91.
[5]	CHENG Jing-wen, CAO Lei, ZHANG Yan-min, YE Qian, CHEN Min, TAN Wen-song, ZHAO Liang. Establishment and Application of Multigene Engineering Transformation Strategy for CHO Cells [J]. Biotechnology Bulletin, 2023, 39(2): 283-291.
[6]	HUANG Wen-li, LI Xiang-xiang, ZHOU Wen-ting, LUO Sha, YAO Wei-jia, MA Jie, ZHANG Fen, SHEN Yu-sen, GU Hong-hui, WANG Jian-sheng, SUN Bo. Targeted Editing of BoZDS in Broccoli by CRISPR/Cas9 Technology [J]. Biotechnology Bulletin, 2023, 39(2): 80-87.
[7]	WANG Bing, ZHAO Hui-na, YU Jing, CHEN Jie, LUO Mei, LEI Bo. Regulation of Leaf Bud by REVOLUTA in Tobacco Based on CRISPR/Cas9 System [J]. Biotechnology Bulletin, 2023, 39(10): 197-208.
[8]	LI Shuang-xi, HUA Jin-lian. Research Progress in Anti-porcine Reproductive and Respiratory Syndrome Genetically Modified Pigs [J]. Biotechnology Bulletin, 2023, 39(10): 50-57.
[9]	LIN Rong, ZHENG Yue-ping, XU Xue-zhen, LI Dan-dan, ZHENG Zhi-fu. Functional Analysis of ACOL8 Gene in the Ethylene Synthesis and Response in Arabidopsis thaliana [J]. Biotechnology Bulletin, 2023, 39(1): 157-165.
[10]	LIU Jing-jing, LIU Xiao-rui, LI Lin, WANG Ying, YANG Hai-yuan, DAI Yi-fan. Establishment of Porcine Fetal Fibroblasts with OXTR-knockout Using CRISPR/Cas9 [J]. Biotechnology Bulletin, 2022, 38(6): 272-278.
[11]	Olalekan Amoo, HU Li-min, ZHAI Yun-gu, FAN Chu-chuan, ZHOU Yong-ming. Regulation of Shoot Branching by BRANCHED1 in Brassica napus Based on Gene Editing Technology [J]. Biotechnology Bulletin, 2022, 38(4): 97-105.
[12]	DING Ya-qun, DING Ning, XIE Shen-min, HUANG Meng-na, ZHANG Yu, ZHANG Qin, JIANG Li. Construction of Vps28 Knock-out Mice and Model Study of the Impact on Lactation and Immune Traits [J]. Biotechnology Bulletin, 2022, 38(3): 164-172.
[13]	YAN Jiong, FENG Chen-yi, GAO Xue-kun, XU Xiang, YANG Jia-min, CHEN Zhao-yang. Construction of Homozygous Plin1-knockout Mouse Model and Phenotype Analysis Based on CRISPR/Cas9 Technology [J]. Biotechnology Bulletin, 2022, 38(3): 173-180.
[14]	LIU Meng-meng, HAN Li-jun, LIU Bao-ling, XUE Jin-ai, LI Run-zhi. Cloning and Expression Analysis of GhSDP1 and Its Promoter in Gossypium hirsutum [J]. Biotechnology Bulletin, 2022, 38(2): 34-43.
[15]	ZHONG Jing, SUN Ling-ling, ZHANG Shu, MENG Yuan, ZHI Yi-fei, TU Li-qing, XU Tian-peng, PU Li-ping, LU Yang-qing. Effect of Knocking Out the Mda5 Gene by CRISPR/Cas9 Technology on the Replication of Newcastle Disease and Infectious Bursal Virus [J]. Biotechnology Bulletin, 2022, 38(11): 90-96.