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Enhance genome editing efficiency and specificity by a quick CRISPR/Cas9 system

Hu, Y.; Luo, Z.; Li, J.; Wang, D.; Sun, H.-X.; Xiao, A.; Liu, D.; Cheng, Z.; Wu, J.; Shen, Y.; Xu, X.; Zhang, B.; Wang, J.; Gu, Y.; Yang, H.

2019-08-02 genetics
10.1101/708404 bioRxiv
Show abstract

CRISPR/Cas9 is a powerful genome editing tool that has been successfully applied to a variety of species, including zebrafish. However, targeting efficiencies vary greatly at different genomic loci, the underlying causes of which were still elusive. Here we report a quick CRISPR/Cas9 system, designated as qCas9, which exhibits accelerated turnover of Cas9 protein in zebrafish. Our data showed that qCas9 significantly improved targeting efficiency, including both knock-out and knock-in in F0 embryos, and yielded higher germline transmission rate in founder screen. Importantly, qCas9 showed little to no off-target editing in zebrafish and profoundly reduced off-target effect in HEK293T cell line. In summary, our findings demonstrate that qCas9 is a simple, economic and highly effective method to improve genome editing efficiency in zebrafish embryos and also holds great potential in reducing off-target effect in mammalian cell lines.

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