Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, P.R. China.
Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, P.R. China.
Mol Plant. 2021 Feb 1;14(2):352-360. doi: 10.1016/j.molp.2020.12.017. Epub 2020 Dec 28.
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.
化脓链球菌 Cas9（SpCas9）是植物中应用最广泛的基因组编辑工具。SpCas9 诱导的编辑严格需要规范的 NGG 前间隔基序（PAM），显著限制了其应用范围。最近，开发了五种 SpCas9 变体，SpCas9-NRRH、SpCas9-NRCH、SpCas9-NRTH、SpG 和 SpRY，以在人类细胞中识别非规范的 PAM。在本研究中，这些变体被用于植物基因组编辑，并通过稳定转化在水稻（Oryza sativa）中各种规范和非规范 PAM 位点上全面检查了它们的靶向突变能力。此外，基于这些 SpCas9 变体开发了使用大鼠 APOBEC1 或人 APOBEC3a 的胞嘧啶碱基编辑器和使用直接进化的高度相容的 TadA∗-8e 脱氨酶的腺嘌呤碱基编辑器。我们的结果表明，开发的基于 SpCas9 变体的碱基编辑器可在具有非规范 PAM 的转基因水稻系中的靶位点上轻易地在 C∙G 和 T∙A 之间进行转换。总的来说，本研究中开发的工具包大大扩展了 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敲除和精确编辑。
