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无细胞系统中CRISPR核糖核蛋白置换的研究

A Study of CRISPR Ribonucleoprotein Displacement in Cell-Free Systems.

作者信息

Smith Randi L, Davenport Peter W, Lakin Matthew R

机构信息

Center for Biomedical Engineering, University of New Mexico, Albuquerque, New Mexico 87131, United States.

Department of Computer Science, University of New Mexico, Albuquerque, New Mexico 87131, United States.

出版信息

ACS Omega. 2025 Feb 26;10(9):9154-9164. doi: 10.1021/acsomega.4c09275. eCollection 2025 Mar 11.

Abstract

CRISPR/Cas-based transcription factors are a powerful tool for controlling gene expression in living cells and cell-free systems, as their programmable DNA-binding activity makes them a powerful tool for building and scaling up engineered genetic networks. The use of guide RNAs for targeting Cas proteins to desired binding sites opens up the possibility of using RNA engineering techniques to achieve programmable and dynamic control of CRISPR/Cas-based transcription factor activity and hence of gene expression. In this work, we investigate the use of RNA strand displacement systems to remove bound CRISPR/Cas ribonucleoprotein complexes from target DNA in cell-free systems. The binding of catalytically inactive dCas9 is monitored by using CRISPR interference to repress the expression of a reporter protein. We express an antisense RNA complementary to an extended toehold on an engineered guide RNA in an -based cell-free expression system with the goal of rapidly removing bound CRISPR/Cas ribonucleoproteins via strand displacement. We find that dCas9 appears to be surprisingly resistant to removal via this mechanism, which indicates that other strategies for dynamic removal of bound Cas proteins may prove to be more effective.

摘要

基于CRISPR/Cas的转录因子是控制活细胞和无细胞系统中基因表达的强大工具,因为其可编程的DNA结合活性使其成为构建和扩大工程遗传网络的有力工具。使用引导RNA将Cas蛋白靶向到所需的结合位点,开启了利用RNA工程技术实现对基于CRISPR/Cas的转录因子活性以及基因表达进行可编程和动态控制的可能性。在这项工作中,我们研究了在无细胞系统中使用RNA链置换系统从靶DNA中去除结合的CRISPR/Cas核糖核蛋白复合物的方法。通过使用CRISPR干扰来抑制报告蛋白的表达,监测催化失活的dCas9的结合情况。我们在基于大肠杆菌的无细胞表达系统中表达与工程化引导RNA上的延伸引发位点互补的反义RNA,目的是通过链置换快速去除结合的CRISPR/Cas核糖核蛋白。我们发现,dCas9似乎对通过这种机制的去除具有惊人的抗性,这表明其他动态去除结合的Cas蛋白的策略可能会被证明更有效。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6182/11904657/847651b3f86f/ao4c09275_0001.jpg

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