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通过扩大CRISPR-Cas9的识别域来提高腺基编辑精度

Shuliang Gao¹, Benson Weng¹, Douglas Wich¹

¹Department of Biomedical Engineering, Tufts University, Medford, MA, USA.

Nature communications

|February 28, 2025

概括

研究人员设计了一个更大的Streptococcus pyogenes Cas9 (SpCas9) 来提高基因编辑精度. 这种修改后的Cas9通过减少意想不到的编辑来改进腺基编辑器,为优化基因编辑工具提供了一个新的策略.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
蛋白质工程是指蛋白质工程.

背景情况:

域扩张是RNA引导内核酶多样化的已知驱动因素.
在Cas9中REC域扩展的具体好处仍然在很大程度上未被探索.
提高像Cas9这样的基因编辑工具的精度对于治疗应用至关重要.

研究的目的:

研究REC域扩张对Streptococcus pyogenes Cas9 (SpCas9) 功能的影响.
为了设计一个具有增强编辑精度的"巨型"SpCas9 (GS-Cas9).
探索扩大REC域在调节结酶的基础编辑中的实用性.

主要方法:

在SpCas9.9中确定了一个适合大型REC域插入的插入位.
通过扩大 REC 域,构建了一个类似自然进化的巨型 SpCas9 (GS-Cas9).
通过调节绑定的TadA8e来评估GS-Cas9的编辑精度及其对腺基编辑器 (ABE8e) 的影响.

主要成果:

成功创建了具有显著扩大非催化REC域的GS-Cas9.
与野生型SpCas9.9相比,GS-Cas9的基因编辑精度大大提高.
扩大REC域允许更好地调节绑定的TadA8e,减少非目标编辑,提高ABE8e精度.

结论:

对Cas9的进化灵感扩展的概念证明,为优化基因编辑器提供了一种新的方法.
扩大REC域是一个可行的策略,可以提高Cas9和基础编辑器的精度.
突出了RNA引导内核酶和基基编辑器的拓灵活性工程的潜力.

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