利用 CRISPR/Cas 介导的碱基编辑器在水稻中进行精确碱基替换。
Precise Base Substitution Using CRISPR/Cas-Mediated Base Editor in Rice.
机构信息
Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
Institute of Fruit Tree and Tea Science, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
出版信息
Methods Mol Biol. 2025;2869:101-111. doi: 10.1007/978-1-0716-4204-7_11.
Base editors, CRISPR/Cas-based precise genome editing tools, enable base conversion at a target site without inducing DNA double-strand breaks. The genome editing targetable range is restricted by the requirement for protospacer adjacent motif (PAM) sequence. Cas9 derived from Streptococcus pyogenes (SpCas9)-most widely used for genome editing in many organisms-requires an NGG sequence adjacent to the target site as a PAM. Then, engineered and natural Cas variants with altered PAM recognition are used for base editor to expand the flexibility of base substitution position. In this chapter, we describe a protocol for base editing based on SpCas9-NG, which is a rationally engineered SpCas9 variant that can recognize relaxed NG PAMs.
碱基编辑器是一种基于 CRISPR/Cas 的精确基因组编辑工具，可在不诱导 DNA 双链断裂的情况下实现靶位点的碱基转换。基因组编辑的靶向范围受到原间隔序列邻近基序（PAM）序列的限制。来源于化脓性链球菌（SpCas9）的 Cas9 是目前在许多生物中用于基因组编辑最广泛的 Cas9，它需要靶位点附近的 NGG 序列作为 PAM。然后，使用经过工程改造的和天然的 Cas 变体来识别改变的 PAM，以用于碱基编辑器来扩展碱基替换位置的灵活性。在本章中，我们描述了一种基于 SpCas9-NG 的碱基编辑方法，SpCas9-NG 是一种经过合理设计的 SpCas9 变体，能够识别宽松的 NG PAMs。
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