Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.
Nat Protoc. 2023 Nov;18(11):3194-3228. doi: 10.1038/s41596-023-00877-w. Epub 2023 Oct 4.
Fusing apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like cytidine deaminase with catalytically impaired Cas proteins (e.g., nCas9 or dCas9) provides a novel gene-editing technology, base editing, that grants targeted base substitutions with high efficiency. However, genome-wide and transcriptome-wide off-target mutations are observed in base editing, which raises safety concerns regarding therapeutic applications. Previously, we developed a new base editing system, the transformer base editor (tBE), to induce efficient editing with no observable genome-wide or transcriptome-wide off-target mutations both in mammalian cells and in mice. Here we describe a detailed protocol for the design and application of the tBE. Steps for designing single-guide RNA (sgRNA) and helper sgRNA pairs, making constructs, determining the genome-wide and transcriptome-wide off-target mutations, producing the tBE-containing adeno-associated viruses, delivering adeno-associated viruses into mice and examining the in vivo editing effects are included in this protocol. High-precision base editing by the tBE can be completed within 2-3 weeks (in mammalian cells) or within 6-8 weeks (in mice), with sgRNA-helper sgRNA pairs. The whole process can be collaboratively accomplished by researchers using standard techniques from molecular biology, bioinformatics and mouse husbandry.
将载脂蛋白 B mRNA 编辑酶催化多肽样胞嘧啶脱氨酶与催化失活的 Cas 蛋白（如 nCas9 或 dCas9）融合提供了一种新的基因编辑技术，碱基编辑，可高效实现靶向碱基替换。然而，碱基编辑中观察到全基因组和转录组范围的脱靶突变，这引发了治疗应用的安全性问题。先前，我们开发了一种新的碱基编辑系统，即变形碱基编辑器（tBE），在哺乳动物细胞和小鼠中均能诱导高效编辑，且没有观察到全基因组或转录组范围的脱靶突变。本研究描述了 tBE 的设计和应用的详细方案。设计单指导 RNA（sgRNA）和辅助 sgRNA 对、构建载体、确定全基因组和转录组范围的脱靶突变、制备含 tBE 的腺相关病毒、将腺相关病毒递送至小鼠并检测体内编辑效果的步骤均包含在本方案中。使用 sgRNA-helper sgRNA 对，tBE 可以在 2-3 周（在哺乳动物细胞中）或 6-8 周（在小鼠中）内完成高精度碱基编辑。整个过程可以由使用分子生物学、生物信息学和小鼠饲养标准技术的研究人员共同完成。
