Xiong Xiangyu, Liu Kehui, Li Zhenxiang, Xia Fan-Nv, Ruan Xue-Ming, He Xionglei, Li Jian-Feng
State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Plant Resources, MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Sun Yat-sen University, Guangzhou, China.
Nat Plants. 2023 Nov;9(11):1832-1847. doi: 10.1038/s41477-023-01540-8. Epub 2023 Oct 16.
Base editors (BEs) empower the efficient installation of beneficial or corrective point mutations in crop and human genomes. However, conventional BEs can induce unpredictable guide RNA (gRNA)-independent off-target edits in the genome and transcriptome due to spurious activities of BE-enclosing deaminases, and current improvements mostly rely on deaminase-specific mutagenesis or exogenous regulators. Here we developed a split deaminase for safe editing (SAFE) system applicable to BEs containing distinct cytidine or adenosine deaminases, with no need of external regulators. In SAFE, a BE was properly split at a deaminase domain embedded inside a Cas9 nickase, simultaneously fragmenting and deactivating both the deaminase and the Cas9 nickase. The gRNA-conditioned BE reassembly conferred robust on-target editing in plant, human and yeast cells, while minimizing both gRNA-independent and gRNA-dependent off-target DNA/RNA edits. SAFE also substantially increased product purity by eliminating indels. Altogether, SAFE provides a generalizable solution for BEs to suppress off-target editing and improve on-target performance.
碱基编辑器（BEs）能够在作物和人类基因组中高效引入有益或校正性点突变。然而，传统的碱基编辑器由于其所含脱氨酶的非特异性活性，会在基因组和转录组中诱导不可预测的、不依赖向导RNA（gRNA）的脱靶编辑，并且目前的改进大多依赖于脱氨酶特异性诱变或外源调节剂。在此，我们开发了一种适用于包含不同胞嘧啶或腺嘌呤脱氨酶的碱基编辑器的分裂脱氨酶安全编辑（SAFE）系统，无需外部调节剂。在SAFE系统中，碱基编辑器在嵌入Cas9切口酶的脱氨酶结构域处进行适当分裂，同时使脱氨酶和Cas9切口酶片段化并失活。gRNA条件下的碱基编辑器重新组装在植物、人类和酵母细胞中实现了强大的靶向编辑，同时将不依赖gRNA和依赖gRNA的脱靶DNA/RNA编辑降至最低。SAFE系统还通过消除插入缺失显著提高了产物纯度。总之，SAFE系统为碱基编辑器提供了一种通用的解决方案，以抑制脱靶编辑并提高靶向编辑性能。
