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xCas9 expands the scope of genome editing with reduced efficiency in rice
Junjie Wang, Xiangbing Meng, Xixun Hu, Tingting Sun, Jiayang Li, Kejian Wang* and Hong Yu*
Plant Biotechnology Journal  , 2019, 17:709-711.

Abstract

Dear Editor,

CRISPR-Cas9 systems have been widely used in animals and plants for genome editing, epigenetic modification, etc. (Cong et al., 2013; Hilton et al., 2015). However, the accurate recognition of target sites of CRISPR/Cas9 systems depends on the single‐guide RNA (sgRNA) corresponding to each target site and protospacer adjacent motifs (PAMs) around these sites (Anders et al., 2014). Cas9‐sgRNA complexes will directly eject the DNA template if no PAMs are recognized before base pairing between target sites and sgRNA (Sternberg et al., 2014). Therefore, PAM recognition is an essential step for the function of Cas9‐sgRNA complexes. The common Streptococcus pyogenes Cas9 (SpCas9) recognizes canonical NGG PAM, which restricts the editable range of the rice genome. In an effort to overcome this limitation, several studies have reported that other Cas effectors (Cpf1 for AT‐rich PAMs) and engineered Cas9 variants (VQR for NGA PAMs and VRER for NGCG PAMs) could be employed with other PAMs for rice genome editing (Hu et al., 2016; Zetsche et al., 2015). In addition, another study showed that SpCas9 could robustly recognize NAG PAMs and cleave target sites in rice genome (Meng et al., 2018). Although several tools have been developed for genome editing, versatile nucleases recognizing various PAMs are still largely required to expand the genome editing toolbox. Recently, xCas9, a Cas9 variant that recognizes most types of PAM reported in mammalian cells, including NG, GAA and GAT, was developed (Hu et al., 2018). However, the efficiency of this system in other species has not been reported yet. Here, we generated two versions of efficient xCas9 variants to expand the scope of genome editing in rice.