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I-F型CAST的结构引导工程用于在人类细胞中向基因插入

George D Lampe¹, Ashley R Liang^1,2, Dennis J Zhang^1,3

¹Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.

bioRxiv : the preprint server for biology

|September 30, 2024

概括

研究人员设计了CRISPR关联转基因酶 (CAST) 变体,以实现精确的双链无断基因组编辑. 这些新的CAST系统提高了DNA集成效率,并为人类基因组工程应用提供了新的工具.

科学领域:

分子生物学分子生物学
基因组学就是基因组学.
生物化学生物化学

背景情况:

传统的基因组编辑依赖于DNA双链断裂 (DSB),导致不可预测的结果.
与CRISPR相关的转基因酶 (CAST) 提供了一种新的方法来实现无DSB的DNA集成.
类型I-F CAST系统PseCAST已经显示出在人体细胞中进行大规模DNA插入的潜力,但其效率受到限制.

研究的目的:

阐明PseCAST QCascade复合体对目标DNA识别的结构基础.
为了设计PseCAST变体,提高DNA整合效率和改变PAM特异性.
设计混合CAST系统,结合增强的DNA结合和集成能力.

主要方法:

单粒子冷电子显微镜 (cryo-EM) 用于确定PseCAST QCascade复合物的结构.
向DNA图书馆屏幕和局部定向突变发生,以确定关键蛋白质-DNA相互作用.
虚拟CAST系统的合理设计和构建.

主要成果:

化EM揭示了PseCAST中的新型亚型特异相互作用和RNA-DNA异重复特征.
工程 CAST 变种显示了更高的集成效率和更改的 PAM 识别.
结构导向设计使得能够创建混合CAST,具有增强的DNA结合和集成模块.

结论:

对PseCAST的结构洞察力为了解I-F型CAST机制提供了基础.
工程 CAST 变体和混合系统代表了无 DSB 基因组编辑的重大进步.
这项工作为开发用于治疗基因组工程的新型RNA引导转移酶架构提供了多种策略.

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