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SaCas9 Requires 5′‐NNGRRT‐3′ PAM for Sufficient Cleavage and Possesses Higher Cleavage Activity than SpCas9 or FnCpf1 in Human Cells
Author(s) -
Xie Haihua,
Tang Lianchao,
He Xiubin,
Liu Xiexie,
Zhou Chenchen,
Liu Junjie,
Ge Xianglian,
Li Jin,
Liu Changbao,
Zhao Junzhao,
Qu Jia,
Song Zongming,
Gu Feng
Publication year - 2018
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201700561
Subject(s) - cas9 , genome editing , crispr , plasmid , endonuclease , biology , computational biology , cleavage (geology) , gene , guide rna , human genome , genome , reporter gene , microbiology and biotechnology , genetics , gene expression , paleontology , fracture (geology)
CRISPR/Cas9‐mediated gene therapy holds great promise for the treatment of human diseases. The protospacer adjacent motif (PAM), the sequence adjacent to the target sequence, is an essential targeting component for the design of CRISPR/Cas9‐mediated gene editing. However, currently, very few studies have attempted to directly study the PAM sequence in human cells. To address this issue, the authors develop a dual fluorescence reporter system that could be harnessed for identifying functional PAMs for genome editing endonuclease, including Cas9. With this system, the authors investigate the effects of different PAM sequences for SaCas9, which is small and has the advantage of allowing in vivo genome editing, and found only 5′‐NNGRRT‐3′ PAM could induced sufficient target cleavage with multi‐sites. The authors also found SaCas9 possesses higher activity than SpCas9 or FnCpf1 via plasmids (episomal) and chromosomes with integrated eGFP‐based comparison. Taken together, the authors show that a dual fluorescence reporter system is a means to identifying a functional PAM and quantitatively comparing the efficiency of different genome editing endonucleases with the similar or identical target sequence in human cells.