慢病毒载体介导稳定表达Cpf1的细胞系的构建及其基因编辑效率验证.

【Objective】This paper aimed to establish the 293 T and HT 1080 cell lines with CRISPR/Cpf1 gene by lentiviral vector and further to realize the high-efficiency gene editing. 【Method】AsCpf1 and LbCpf1 gene was amplified by PCR technique, which was inserted into the pLent-EF1 a-MF-CMV-GFP-P2 A-Puro vector. Monolayer of 293 FT cells were cotransfected with three plasmids psPAX2, pMD2.G and Cpf1 in pLent-EF1 a-MF-CMV-GFP-P2 A-Puro. The recombinant lentivirus expressing LbCpf1 and AsCpf1 was harvested, and the titer of virus was calculated by fluorescence microscope 48 hours later. The purified lentivirus was used to infect 293 T cell and the transduced cells were selected with puromycin and single cell colonies were isolated and verified for DNA, RNA and cDNA. Single cell colonies were transfected by sgRNA targeting the exons ABCG1 CDS regions to the functional domains of ABCG1 genes, and gene knockout efficiency was measured by DNA specificity and T7 EΙ digestion. 【Result】DNA sequnencing verified that Cpf1-pLent was constructed successfully, and the titer of recombinant lentivirus was greater than 10~6 TU/mL. Three cell lines expressing Cpf1 stably were obtained, two of which(AsCpf1-293 T-7 D and AsCpf1-HT1080 02-3 B) the targeted gene was knocked out successfully by T7 EΙ digestion. 【Conclusion】 293 T and HT 1080 cells stably expressing Cpf1 was successfully constructed, which could be a valuable tool for gene editing. [ABSTRACT FROM AUTHOR]