Streptococcus pyogenes Cas9 (SpCas9) is the most widely used genome editing tool in plants. The editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif (PAM), significantly limiting its scope of application. Recently, five SpCas9 variants, SpCas9-NRRH, SpCas9-NRCH, SpCas9-NRTH, SpG, and SpRY, were developed to recognize non-canonical PAMs in human cells. In this study, these variants were engineered for plant genome editing, and their targeted mutagenesis capabilities were comprehensively examined at various canonical and non-canonical PAM sites in rice (Oryza sativa) by stable transformation. Moreover, both cytosine base editors using a rat APOBEC1 or a human APOBEC3a and adenine base editors using a directly evolved highly compatible TadA∗-8e deaminase were developed from these SpCas9 variants. Our results demonstrated that the developed SpCas9 variants-based base editors readily generated conversions between C∙G and T∙A in the target sites with non-canonical PAMs in transgenic rice lines. Collectively, the toolbox developed in this study substantially expands the scope of SpCas9-mediated genome editing and will greatly facilitate gene disruption and precise editing in plants.

化脓性链球菌Cas9（SpCas9）是植物中使用最广泛的基因组编辑工具。SpCas9诱导的编辑严格要求使用规范的NGG原型间隔子相邻主题（PAM），从而极大地限制了其应用范围。最近，开发了五种SpCas9变体，即SpCas9-NRRH，SpCas9-NRCH，SpCas9-NRTH，SpG和SpRY，以识别人细胞中的非规范PAM。在这项研究中，这些变种被设计用于植物基因组编辑，并在水稻各种经典和非经典PAM网站进行了全面检查了他们的定向诱变能力（水稻）通过稳定的转化。而且，从大鼠SpOB9或人APOBEC3a的胞嘧啶碱基编辑器和使用直接进化的高度兼容的TadA * -8e脱氨酶的腺嘌呤碱基编辑器都是从这些SpCas9变异体开发出来的。我们的结果表明，开发的基于SpCas9变体的基础编辑器可以通过转基因水稻品系中的非经典PAM在目标位点轻松实现C∙G和T∙A之间的转换。总体而言，本研究开发的工具箱极大地扩展了SpCas9介导的基因组编辑的范围，并将极大地促进植物中的基因破坏和精确编辑。