PT  - JOURNAL ARTICLE
AU  - Shan-Ye Gu
AU  - Jia Li
AU  - Jian-Bin Cao
AU  - Ji-Wen Bu
AU  - Yong-Gang Ren
AU  - Wen-Jie Du
AU  - Zhe-Cong Chen
AU  - Chu-Fan Xu
AU  - Min-Cang Wang
AU  - Lai Jiang
AU  - Cheng Huang
AU  - Jiu-Lin Du
TI  - Efficient replacement of long DNA fragments via non-homologous end joining at non-doding regions
AID  - 10.1101/2020.01.11.902791
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.01.11.902791
4099  - http://biorxiv.org/content/early/2020/01/14/2020.01.11.902791.short
4100  - http://biorxiv.org/content/early/2020/01/14/2020.01.11.902791.full
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.