Construction of recombinant plasmids with the clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (Cas9) system

RH Rong-Quan He
JL Jian-Di Li
XD Xiu-Fang Du
YD Yi-Wu Dang
LY Lin-Jie Yang
ZH Zhi-Guang Huang
LL Li-Min Liu
LL Liu-Feng Liao
HY Hong Yang
GC Gang Chen
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We designed two gRNA sequences gRNA1: 5′-GGCGTCGAAGTAGGACGAGTG-3′ and gRNA2: 5′-GTCTGACCGGGACACGAACAC-3′. By synthesizing the oligo-DNAs of these gRNAs and annealing them, then cloning them into backbone vectors ‘pSpCas9(BB)-2A-Puro (PX459) V2.0’ (Plasmid #62988, addgene), two gRNA-expressing plasmids were formed [32].

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