State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou, China.
Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China.
Plant Biotechnol J. 2022 May;20(5):934-943. doi: 10.1111/pbi.13774. Epub 2022 Jan 17.
Adenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site-specific A-to-G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer-adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high-efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single-stranded DNA-binding domain (DBD), based on PAM-less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n-NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base-editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e-DBD-SpRYn (hyABE8e-SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)-dependent off-target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base-editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.
腺嘌呤碱基编辑器(ABEs),通常是经过工程改造的腺嘌呤脱氨酶和 Cas 变体,可在特定位置引入 A 到 G 的突变,从而改善农艺性状。然而,编辑效率显著变化、对邻近基序(PAMs)的严格要求以及狭窄的编辑窗口极大地限制了它们的应用。在这里,我们通过融合一种经过进化的、高度活跃的腺嘌呤脱氨酶 TadA8e 和一种单链 DNA 结合结构域(DBD),开发了一个用于植物的强大高效 ABE(PhieABE)工具箱,该工具箱基于无 PAM/自由链球菌 Cas9(SpCas9)切口酶变体,可识别 PAM NGN(用于 SpCas9n-NG 和 SpGn)或 NNN(用于 SpRYn)。通过靶向水稻中的 29 个代表性靶标并评估结果,我们证明 PhieABEs 与 ABE7.10 和通用 ABE8e 系统相比,具有显著提高的碱基编辑活性、扩大的靶标范围和更宽的编辑窗口。在这些 PhieABEs 中,超 ABE8e-DBD-SpRYn(hyABE8e-SpRY)在一些测试位点的编辑效率接近 100%,编辑窗口中具有高比例的纯合碱基替换,并且没有 sgRNA 依赖性脱靶变化。原始 sgRNA 比进化后的 sgRNA 更兼容 PhieABEs。总之,DBD 融合有效地提高了碱基编辑效率,这个新的 PhieABE 工具箱应该在植物功能基因组学和作物改良中有广泛的应用。