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在真核细胞中设计了与转录相关的Cas9向

Gregory W Goldberg¹, Manjunatha Kogenaru¹, Sarah Keegan¹

¹Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY 10016, USA.

bioRxiv : the preprint server for biology

|October 2, 2023

概括

工程CRISPR-Cas9系统通过将其与活跃转录联系起来来实现对DNA向的时间控制. 这种转录相关的Cas9向 (TraCT) 系统可以在酵母和人类细胞中精确编辑基因.

科学领域:

分子生物学分子生物学
基因编辑基因编辑
生物技术是生物技术.

背景情况:

2类CRISPR-Cas效应核酶,如Cas9,利用原体空间相邻动机 (PAM) 并指导RNA相互作用用于DNA向.
这些相互作用提供了空间控制,而不是时间控制,导致构成性DNA结合和分裂.
现有的CRISPR-Cas系统缺乏固有的机制来调节DNA向的时间调节.

研究的目的:

设计一个具有对DNA定位的时空控制的CRISPR-Cas9系统,将其与活性转录相合.
为了证明转录相关的Cas9向 (TraCT) 在真核细胞中的可行性.
探索TraCT用于选择性基因编辑的应用.

主要方法:

设计的Cas9融合蛋白旨在将新生RNA与目标DNA序列附近结合.
利用低于最佳的PAM相互作用来限制基底Cas9活性.
在真核酵母和人类细胞中测试了TraCT系统,并将其应用于选择性基因编辑场景.

主要成果:

使用工程 Cas9 融合蛋白 (TraCT) 证明了转录和 DNA 向之间的成功时空合.
在酵母和人体细胞中,当不理想的PAM相互作用存在并且新生RNA与DNA结合时,TraCT就能起作用.
在不同的基因位点中实现了对相同目标的选择性编辑,并在酵母菌中实现了差异转录的等位基因位点.

结论:

对Cas9向的时间控制可以在不改变核心Cas9域或引导RNA组件的情况下进行工程设计.
同转录RNA结合作为一个cis作用机制,有条件地激活CRISPR-CasDNA向.
在真核生物系统中,TraCT系统提供了一种用于精确和调节基因编辑的新方法.

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