Expanding base editing scope to near-PAMless with engineered CRISPR/Cas9 variants in plants

摘要

Dear Editor, Base editors (BEs), including cytosine base editor (CBE) and adenine base editor (ABE), have been widely used to generate irreversible nucleotide substitution in plants and animals.However, their wide applications are largely hindered by the strict NG protospacer adjacent motif (PAM) sequences recognized by Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants, such as SpCas9-NG and xCas9 (Hua et al., 2019;Ren et al.,2019;Wu et al., 2019;Zhong et al., 2019;Zhang et al., 2020).Most recently, it was reported that three new SpCas9 variants,SpCas9-NRRH, SpCas9-NRTH, and SpCas9-NRCH, could recognize non-G PAMs (NRNH, where R is A or G and H is A,C, or T) in human cells (Miller et al., 2020).Meanwhile, SPRY,another new SpCas9 variant, was developed to greatly expand the editing scope of BEs to nearly PAMless (Walton et al.,2020).In this study, we generated a series of efficient BE toolkits and almost achieved C-to-T mutation without PAM restriction except for NTG PAM, and largely expanded A-to-G mutation scope in stable transformed rice, providing a reference for application in other plants.