Zhang Yaqin, Chen Jianai, Wu Zhina, Zhao Chenfei, Wang Rui, Li Zhiping, Wang Jiasi, Wang Di
Department of Clinical Pharmacy, The First Hospital of Jilin University, Jilin University, Changchun, Jilin, 130021, China.
School of Life Sciences, Jilin University, Changchun, Jilin, 130012, China.
Adv Sci (Weinh). 2025 Jan;12(3):e2407194. doi: 10.1002/advs.202407194. Epub 2024 Nov 22.
The clustered regularly interspaced palindromic repeats (CRISPR) /CRISPR-associated proteins (Cas) system is the immune system in bacteria and archaea and has been extensively applied as a critical tool in bioengineering. Investigation of the mechanisms of catalysis of CRISPR/Cas systems in intracellular environments is essential for understanding the underlying catalytic mechanisms and advancing CRISPR-based technologies. Here, the catalysis mechanisms of CRISPR/Cas systems are investigated in an aqueous two-phase system (ATPS) comprising PEG and dextran, which simulated the intracellular environment. The findings revealed that nucleic acids and proteins tended to be distributed in the dextran-rich phase. The results demonstrated that the cis-cleavage activity of Cas12a is enhanced in the ATPS, while its trans-cleavage activity is suppressed, and this finding is further validated using Cas13a. Further analysis by increasing the concentration of the DNA reporter revealed that this phenomenon is not attributed to the slow diffusion of the reporter, and explained why Cas12a and Cas13a do not randomly cleave nucleic acids in the intracellular compartment. The study provides novel insights into the catalytic mechanisms of CRISPR/Cas systems under physiological conditions and may contribute to the development of CRISPR-based molecular biological tools.
成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）系统是细菌和古生菌中的免疫系统，并已被广泛用作生物工程中的关键工具。研究CRISPR/Cas系统在细胞内环境中的催化机制对于理解潜在的催化机制和推动基于CRISPR的技术至关重要。在此，在由聚乙二醇（PEG）和葡聚糖组成的模拟细胞内环境的双水相系统（ATPS）中研究了CRISPR/Cas系统的催化机制。研究结果表明，核酸和蛋白质倾向于分布在富含葡聚糖的相中。结果表明，Cas12a的顺式切割活性在ATPS中增强，而其反式切割活性受到抑制，并且使用Cas13a进一步验证了这一发现。通过增加DNA报告分子的浓度进行的进一步分析表明，这种现象并非归因于报告分子的缓慢扩散，并解释了为什么Cas12a和Cas13a不会在细胞内区室中随机切割核酸。该研究为CRISPR/Cas系统在生理条件下的催化机制提供了新的见解，并可能有助于基于CRISPR的分子生物学工具的开发。
