RT Journal Article
SR Electronic
T1 Efficient replacement of long DNA fragments via non-homologous end joining at non-doding regions
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.01.11.902791
DO 10.1101/2020.01.11.902791
A1 Shan-Ye Gu
A1 Jia Li
A1 Jian-Bin Cao
A1 Ji-Wen Bu
A1 Yong-Gang Ren
A1 Wen-Jie Du
A1 Zhe-Cong Chen
A1 Chu-Fan Xu
A1 Min-Cang Wang
A1 Lai Jiang
A1 Cheng Huang
A1 Jiu-Lin Du
YR 2020
UL http://biorxiv.org/content/early/2020/01/14/2020.01.11.902791.abstract
AB Genomic DNA replacement for achieving sophisticated genetic manipulation is implemented currently through homogenous recombination/homology-dependent repair (HR/HDR). Here we report an efficient DNA fragment replacement method that is mediated by non-homologous end joining (NHEJ)-dependent DNA repair at two sites of CRISPR/Cas9-induced double-strand breaks at non-coding genomic regions flanking the exons of targeted genes. We demonstrated this method by generating three conditional alleles and two reporter lines of zebrafish. Functional assays of the conditional alleles proved that the genomic sequence between two inserted loxP sites was deleted by the Cre recombinase, and the phenotype after Cre-induced excision was comparable to previously reported mutants or morphants. Furthermore, combining double-fluorescence expression donor vectors, we showed that the efficiency of this NHEJ-mediated DNA replacement was around 3 times larger than that of HR/HDR-mediated approach. Our method provides a feasible strategy for genomic DNA replacement in zebrafish, which can be applicable for other organisms as well.