Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Str., 32, 119991, Moscow, Russia.
Department of Biology, Lomonosov Moscow State University, 119234, Moscow, Russia.
Appl Microbiol Biotechnol. 2024 Feb 14;108(1):209. doi: 10.1007/s00253-024-13056-y.
The Cas9 endonuclease of the CRISPR/Cas type IIA system from Streptococcus pyogenes is the heart of genome editing technology that can be used to treat human genetic and viral diseases. Despite its large size and other drawbacks, S. pyogenes Cas9 remains the most widely used genome editor. A vast amount of research is aimed at improving Cas9 as a promising genetic therapy. Strategies include directed evolution of the Cas9 protein, rational design, and domain swapping. The first generation of Cas9 editors comes directly from the wild-type protein. The next generation is obtained by combining mutations from the first-generation variants, adding new mutations to them, or refining mutations. This review summarizes and discusses recent advances and ways in the creation of next-generation genomic editors derived from S. pyogenes Cas9. KEY POINTS: • The next-generation Cas9-based editors are more active than in the first one. • PAM-relaxed variants of Cas9 are improved by increased specificity and activity. • Less mutagenic and immunogenic variants of Cas9 are created.
CRISPR/Cas 型 IIA 系统的 Cas9 内切酶来自酿脓链球菌，是基因组编辑技术的核心，可用于治疗人类遗传和病毒疾病。尽管 S. pyogenes Cas9 体积庞大且存在其他缺点，但它仍是应用最广泛的基因组编辑工具。大量研究旨在改进 Cas9 作为一种有前途的基因治疗方法。策略包括 Cas9 蛋白的定向进化、合理设计和结构域交换。第一代 Cas9 编辑器直接来自野生型蛋白。下一代 Cas9 编辑器通过结合第一代变体中的突变、向其中添加新突变或改进突变而获得。本文总结和讨论了源自 S. pyogenes Cas9 的新一代基因组编辑工具的最新进展和创建方法。关键点：• 基于下一代 Cas9 的编辑器比第一代更活跃。• PAM 松弛的 Cas9 变体通过提高特异性和活性得到改善。• 产生了突变较少且免疫原性较低的 Cas9 变体。
