Two recently engineered SpCas9 variants, namely xCas9 and Cas9-NG, show promising potential in 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 xCas9-3.7, the most effective xCas9 variant in mammalian cells, for targeted mutagenesis at 16 possible NGN PAM (protospacer adjacent motif) combinations in duplicates. xCas9 exhibited nearly equivalent editing efficiency to wild-type Cas9 (Cas9-WT) at most canonical NGG PAM sites tested, whereas it showed 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 that xCas9 had improved targeting specificity over the Cas9-WT. Furthermore, we tested two Cas9-NG variants, Cas9-NGv1 and Cas9-NG, for targeting NGN PAMs. Both Cas9-NG variants showed higher editing efficiency at most non-canonical NG PAM sites tested, and enabled much more efficient editing than xCas9 at AT-rich PAM sites such as GAT, GAA, and CAA. Nevertheless, we found that Cas9-NG variants showed significant reduced activity at the canonical NGG PAM sites. In stable transgenic rice lines, we demonstrated that Cas9-NG had much higher editing efficiency than Cas9-NGv1 and xCas9 at NG PAM sites. To expand the 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.

两个最近设计的SpCas9变体，即xCas9和Cas9-NG，在改善靶向特异性和扩大靶向范围方面显示出有希望的潜力。在这项研究中，我们在模型和农作物水稻中评估了这些Cas9变体。我们首先测试了xCas9-3.7（哺乳动物细胞中最有效的xCas9变体）是否有针对性诱变，重复进行了16种可能的NGN PAM（原间隔子相邻基序）组合。在大多数经测试的规范NGG PAM位点，xCas9表现出与野生型Cas9（Cas9-WT）几乎相同的编辑效率，而在非规范NGH（H = A，C，T）PAM位点，xCas9的活性却有限。通过rAPOBEC1和PmCDA1胞嘧啶脱氨酶的C到T碱基编辑，进一步证明了在NGG PAM上xCas9的高编辑效率。对于错配的sgRNA，我们发现与Cas9-WT相比，xCas9具有更高的靶向特异性。此外，我们测试了两个针对NGN PAM的Cas9-NG变体Cas9-NGv1和Cas9-NG。在大多数经过测试的非规范NG PAM站点上，这两种Cas9-NG变体都显示出更高的编辑效率，并且在富含AT的PAM站点（如GAT，GAA和CAA）上，与xCas9相比，其编辑效率更高。尽管如此，我们发现Cas9-NG变体在规范的NGG PAM位点显示出明显降低的活性。在稳定的转基因水稻品系中，我们证明了在NG PAM位点，Cas9-NG具有比Cas9-NGv1和xCas9更高的编辑效率。为了扩大基础编辑范围，我们通过融合Cas9-NG切口酶（D10A版），PmCDA1和UGI，开发了一种有效的C到T基础编辑系统。在一起