靶向食蟹猴NTCP基因的CRISPR/Cas9系统gRNA筛选

doi:10.16431/j.cnki.1671-7236.2016.10.010

Abstract

Abstract:

This study was aimed to screen gRNA of efficient knockout activity targeting Macaca fascicularis NTCP gene by the CRISPR/Cas9 enzyme digestion method and PCR amplification methods in vitro.Comparing NTCP gene sequences between Macaca fascicularis and human,the sequences of NTCP gene coding amino acid 84 to 87 and 157 to 165 were chosen as gene knockout targets.3 to 4 candidate gRNA sequences were designed in two target sequence regions through gRNA software.By screening cleavage activity targeting NTCP gene in vitro,gRNA1.2 and gRNA2.1 were selected and inserted into pLV hUbC-Cas9-T2A-GFP plasmid,respectively.The genome DNA was extracted from primary hepatocytes after gRNA1.2 and gRNA2.1 being transferred,respectively.Then NTCP sequences were amplified by PCR and sequenced by being cloned into T vector.The results indicated that compared to gRNA1.2,gRNA2.1 had much higher activity to make a frame-shift mutation in NTCP gene.This study laid a theoretical foundation for further editing NTCP gene and its biological function in Macaca fascicularis.

Key words: CRISPR/Cas9; gRNA; Macaca fascicularis; NTCP gene; gene knockout

CLC Number:

XU Gui-li, GAO Xin, LIU Zheng-zhu, GONG Yuan-fang, SONG Hai-feng. gRNA Screening of CRISPR/Cas9 System Targeting Macaca fascicularis NTCP Gene[J]. , 2016, 43(10): 2572-2577.

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gRNA Screening of CRISPR/Cas9 System Targeting Macaca fascicularis NTCP Gene

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