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2018 Khorana Scholar : Dibin Baby Co-Authors Paper

2018 Khorana Scholar Dibin Baby Co-Authors a Paper at University of Maryland

Improving plant genome editing with high-fidelity xCas9 and non-canonical PAM-targeting Cas9-NG

Abstract

Engineered SpCas9 variants, namely xCas9 and Cas9-NG, have shown promising potential on improving targeting specificity and broadening the targeting range. In this study, we evaluated these Cas9 variants in the model and crop plant, rice. We first tested xCas9 (xCas9-3.7, the most effective xCas9 variant in mammalian cells), for targeted mutagenesis at all 16 possible NGN PAM combinations in duplicates. xCas9 mostly exhibited equivalent editing efficiency to wild-type Cas9 (Cas9-WT) at the canonical NGG PAM sites, while possessing limited activity at non-canonical NGH (H=A, C, T) PAM sites. High editing efficiency of xCas9 at NGG PAMs was further demonstrated with C to T base editing by both rAPOBEC1 and PmCDA1 cytidine deaminases. With mismatched sgRNAs, we found xCas9 had improved targeting specificity over the Cas9-WT. We next tested two Cas9-NG variants, Cas9-NGv1 and Cas9-NG, for targeting NGN PAMs. Both Cas9-NG variants showed higher editing efficiency at many non-canonical NG PAM sites. They also enabled efficient editing, much more so than xCas9, at AT-rich PAM sites such as GAT, GAA and CAA. Cas9-NG variants, however, showed significant reduced activity at the canonical NGG PAM sites. In stable transgenic rice lines, we demonstrated Cas9-NG had much higher editing efficiency than Cas9-NGv1 and xCas9 at editing NG PAM sites. To expand base editing scope, we developed an efficient C to T base editing system by making fusion of Cas9-NG nickase (D10A version), PmCDA1 and UGI. Taken together, our work benchmarked xCas9 as a high-fidelity nuclease for targeting canonical NGG PAMs and Cas9-NG as a preferred variant for targeting relaxed PAMs for plant genome editing.

Please view Dibin’s paper here: Improving plant genome editing with high-fidelity xCas9 and non-canonical PAM-trageting Cas9-NG

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