利用CRISPR/Cas9技术敲除RNA甲基化酶TRDMT1基因及其功能初探  

Knockdown of the RNA methyltransferase TRDMT1 gene and its preliminary function study by CRISPR/Cas9 technique

作　　者：徐慧 孙振[1] 薛松磊[1] 豆春峰 胡序明[1] 崔恒宓[1,2] XU Hui;SUN Zhen;XUE Songlei;DOU Chunfeng;HU Xuming;CUI Hengmi(Epigenetics and Epigenetic Institute of Genomics/College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,China;Joint International Research Laboratory of Agriculture ＆ Agri-Product Safety,Ministry of Education,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学动物科学与技术学院/表观遗传学及表观基因组学研究所,江苏扬州225009 [2]扬州大学教育部农业与农产品安全国际合作联合实验室,江苏扬州225009

出　　处：《扬州大学学报（农业与生命科学版）》2018年第2期80-86,共7页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家自然科学基金重大研究计划项目(91540117);国家自然科学基金面上项目(81773013、81372237);国家自然科学基金青年基金项目(31602032);江苏省高校优势学科建设工程项目(2014lO)

摘　　要：为构建TRDMT1基因敲除的HEK293细胞系,利用CRISPR/Cas9系统在TRDMT1基因第1个外显子前插入BGH Ploy(A)转录终止序列,终止TRDMT1基因表达。利用重叠PCR方法合成sgRNA序列,构建TRDMT1-gRNA载体及含不同抗生素基因的同源重组载体TRDMT1-LOXN-Puro和TRDMT1-LOXP-Neo;将同源重组载体TRDMT1-LOXN-Puro、TRDMT1-LOXP-Neo及TRDMT1-gRNA、Cas9表达载体MLM3613共转染HEK293细胞;利用Puromycin和Neomycin抗生素筛选细胞,通过RT-qPCR检测TRDMT1基因转录终止效率。通过细胞生长曲线和划痕试验检测TRDMT1基因敲除对HEK293细胞增殖和迁移能力的影响。结果表明:成功构建了TRDMT1-gRNA载体及TRDMT1-LOXN-Puro、TRDMT1-LOXP-Neo同源重组载体;与正常HEK293细胞相比,试验组TRDMT1表达量仅为1%;TRDMT1基因敲除后显著影响HEK293细胞增殖和迁移。这一研究通过CRISPR/Cas9技术成功构建了TRDMT1基因敲除的HEK293细胞系(HEK293-TKO),为RNA甲基化研究提供了一种有效的工具;TRDMT1基因可能与细胞的增殖和迁移有关。In order to construct the TRDMT1 knockout HEK293 cell line, the BGH Ploy （A） transcriptional terminating sequence was inserted before the first exons of TRDMT1 by the CRISPR/Cas9 system to terminate the expression of the TRDMT1 gene. The gRNA sequences were synthesized and annealed to construct the TRDMTI-gRNA vector. Ho mologous recombinant vectors with two different antibiotic genes were constructed and named as TRDMT1 LOXN-Puro and TRDMT1-LOXP-Neo. The homologous recombinant vectors TRDMT1-LOXN Puro and TRDMT1-LOXP-Neo, TRDMTI-gRNA vector, and Cas9 expression vector MLM3613 were co-transfected into HEK293 cells. The transfected HEK293 cells were selected with Puromycin and Neomycin antibiotics. Transcriptional termination efficiency of TRD- MT1 was evaluated using RT-qPCR. The effects of TRDMT1 deficiency on the proliferation and migration of HEK293 were assessed using cell count method and wound healing assay. The results showed that TRDMTI-gRNA vector and ho- mologous recombination vectors of TRDMT1-LOXN-Puro and TRDMT1-LOXP-Neo were successfully constructed. Com- pared with normal HEK293 cells, the expression level of TRDMT1 in the experimental group was only 1 %. Knockout of TRDMT1 significantly affected the proliferation and migration of HEK293 cells. This study has successfully constructedthe TRDMT1 gene knockout HEK293 ceil line （HEK293-TKO） by CRISPR/Cas9 technology, which provides an effective tool for RNA methylation research. The TRDMT1 gene may be associated with proliferation and migration of cells.

关 键 词：HEK293细胞系 RNA甲基化 m5C TRDMTl基因 CRISPR/Cas9 

分 类 号：Q78[生物学—分子生物学]