A Study of CRISPR Ribonucleoprotein Displacement in Cell-Free Systems

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 E. coli-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.基于CRISPR/Cas的转录因子（CRISPR/Cas-based transcription factors）是调控活细胞与无细胞系统中基因表达的强效工具，其具备可编程的DNA结合活性，可用于构建并规模化改造工程化遗传网络。借助向导RNA（guide RNAs）可将Cas蛋白靶向至预设结合位点，这为利用RNA工程技术实现对基于CRISPR/Cas的转录因子活性乃至基因表达的可编程、动态调控开辟了可行路径。本研究探讨了在无细胞系统中利用RNA链置换系统从靶DNA上移除结合态CRISPR/Cas核糖核蛋白复合物的方法。通过CRISPR干扰（CRISPR interference）抑制报告蛋白（reporter protein）的表达，以此监测催化失活Cas9（dCas9）的结合状态。我们在基于大肠杆菌（E. coli）的无细胞表达系统中，表达一段与工程化向导RNA上延伸的锚定序列（toehold）互补的反义RNA，以期通过链置换机制快速移除结合态CRISPR/Cas核糖核蛋白复合物。实验结果显示，dCas9对此移除机制表现出出人意料的抗性，这表明开发其他用于动态移除结合态Cas蛋白的策略或许会更为有效。