CRISPR/Cas Enzyme Catalysis in Liquid–Liquid Phase‐Separated Systems
Abstract 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...
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| Format: | Article |
| Language: | English |
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Wiley
2025-01-01
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| Series: | Advanced Science |
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| Online Access: | https://doi.org/10.1002/advs.202407194 |
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| _version_ | 1832593529475432448 |
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| author | Yaqin Zhang Jianai Chen Zhina Wu Chenfei Zhao Rui Wang Zhiping Li Jiasi Wang Di Wang |
| author_facet | Yaqin Zhang Jianai Chen Zhina Wu Chenfei Zhao Rui Wang Zhiping Li Jiasi Wang Di Wang |
| author_sort | Yaqin Zhang |
| collection | DOAJ |
| description | Abstract 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. |
| format | Article |
| id | doaj-art-2a66ae3347414a129f693a6c065d5d84 |
| institution | Kabale University |
| issn | 2198-3844 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Advanced Science |
| spelling | doaj-art-2a66ae3347414a129f693a6c065d5d842025-01-20T13:04:18ZengWileyAdvanced Science2198-38442025-01-01123n/an/a10.1002/advs.202407194CRISPR/Cas Enzyme Catalysis in Liquid–Liquid Phase‐Separated SystemsYaqin Zhang0Jianai Chen1Zhina Wu2Chenfei Zhao3Rui Wang4Zhiping Li5Jiasi Wang6Di Wang7Department of Clinical Pharmacy The First Hospital of Jilin University Jilin University Changchun Jilin 130021 ChinaSchool of Life Sciences Jilin University Changchun Jilin 130012 ChinaJilin Provincial Key Laboratory of Tooth Development and Bone Remodeling Department of Orthodontics Hospital of Stomatology Jilin University Changchun 130021 ChinaSchool of Life Sciences Jilin University Changchun Jilin 130012 ChinaDepartment of Physics and Astronomy University of Manchester Manchester M13 9PL UKDepartment of Clinical Pharmacy The First Hospital of Jilin University Jilin University Changchun Jilin 130021 ChinaGuangdong Provincial Key Laboratory of Sensor Technology and Biomedical Instrument School of Biomedical Engineering Shenzhen Campus of Sun Yat‐sen University Shenzhen Guangdong 518107 ChinaSchool of Life Sciences Jilin University Changchun Jilin 130012 ChinaAbstract 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.https://doi.org/10.1002/advs.202407194ATPSCas12aCas13aCRISPR |
| spellingShingle | Yaqin Zhang Jianai Chen Zhina Wu Chenfei Zhao Rui Wang Zhiping Li Jiasi Wang Di Wang CRISPR/Cas Enzyme Catalysis in Liquid–Liquid Phase‐Separated Systems Advanced Science ATPS Cas12a Cas13a CRISPR |
| title | CRISPR/Cas Enzyme Catalysis in Liquid–Liquid Phase‐Separated Systems |
| title_full | CRISPR/Cas Enzyme Catalysis in Liquid–Liquid Phase‐Separated Systems |
| title_fullStr | CRISPR/Cas Enzyme Catalysis in Liquid–Liquid Phase‐Separated Systems |
| title_full_unstemmed | CRISPR/Cas Enzyme Catalysis in Liquid–Liquid Phase‐Separated Systems |
| title_short | CRISPR/Cas Enzyme Catalysis in Liquid–Liquid Phase‐Separated Systems |
| title_sort | crispr cas enzyme catalysis in liquid liquid phase separated systems |
| topic | ATPS Cas12a Cas13a CRISPR |
| url | https://doi.org/10.1002/advs.202407194 |
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