CRISPR/Cas13d介导猪胎儿成纤维细胞SUV39H1/SUV39H2基因敲降

doi:10.16431/j.cnki.1671-7236.2021.06.008

Abstract

Abstract: This study was aimed to knockdown the embryonic development related genes SUV39H1/SUV39H2 in porcine embryonic fibroblasts (PEFs) by CRISPR/Cas13d system,so as to establish a CRISPR/Cas13d mediated gene knockdown system in pigs.According to the coding sequences of SUV39H1/SUV39H2 genes,3 sgRNAs were designed and synthesized in the form of single stranded oligonucleotides.After annealing,they were connected with the sgRNA expression vector linearized by BSPQ Ⅰ.The sgRNA-expressing vectors of SUV39H1 and SUV39H2 genes sgRNA were constructed and sequenced by Sanger software.The expression vector of Cas13d and sgRNA vector targeting SUV39H1/SUV39H2 genes were transfected into PEFs at the ratio of 1∶1,1∶2,1∶4,2∶1 and 4∶1.After 48 h,the cells were collected,and the transfection efficiency was detected by flow cytometry.The knockdown efficiency was detected by semi-quantitative PCR and Real-time quantitative PCR.Semi-quantitative PCR and immunofluorescence were used to detect the level of target gene transcripts and histone H3K9me3 after SUV39H1/SUV39H2 genes knockdown.Sequencing results showed that 3 sgRNAs for each of two genes were successfully inserted into the vector.Flow cytometry results showed that the transfection efficiency was about 70%.Semi-quantitative PCR results showed that 3 sgRNAs extremely significantly reduced the expression of SUV39H1/SUV39H2 genes compared with control group (P<0.01).SUV39H1-sgRNA-2 and SUV39H2-sgRNA-1 could reduce the expression of SUV39H1 and SUV39H2 by 50%.The survival rate of Cas13d∶sgRNA in 1∶1,1∶2 and 1∶4 groups were higher than that of 2∶1 and 4∶1 groups.The knockdown efficiency of Cas13d∶sgRNA in 1∶2,1∶4,2∶1 and 4∶1 groups were significantly higher than that of 1∶1 group (P<0.05),and the knockdown efficiency of Cas13d∶sgRNA in 1∶2 group was the highest (70%).Semi-quantitative PCR results showed that transfection of SUV39H1-sgRNA-2 and SUV39H2-sgRNA-1 extremely significantly reduced the expression of SUV39H1 and SUV39H2,and the expression level was 25%-30% of control group (P<0.01).The results of immunofluorescence showed that the level of histone H3K9me3 decreased significantly after SUV39H1 and SUV39H2 genes knockdown (P<0.05).Therefore,in this study,CRISPR/Cas13d system was used to knockdown SUV39H1 and SUV39H2 successfully,and downregulate H3K9me3 level catalyzed by them.

Key words: CRISPR/Cas13d; SUV39H1/SUV39H2 genes; knockdown; porcine embryonic fibroblasts

CLC Number:

Q786

BI Dengfeng, WANG Yu, YAO Jing, ZHAO Jianguo. CRISPR/Cas13d-mediated SUV39H1/SUV39H2 Gene Knockdown in Porcine Embryonic Fibroblasts[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(6): 1957-1966.

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CRISPR/Cas13d-mediated SUV39H1/SUV39H2 Gene Knockdown in Porcine Embryonic Fibroblasts

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