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

By Yingying Hu, Zhou Luo, Jing Li, Dan Wang, Hai-Xi Sun, An Xiao, Da Liu, Zhenchao Cheng, Jun Wu, Yue Shen, Xun Xu, Bo Zhang, Jian Wang, Ying Gu, Huanming Yang

Posted 02 Aug 2019
bioRxiv DOI: 10.1101/708404

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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