دورية أكاديمية
CRISPR-Cas12a exhibits metal-dependent specificity switching.
| العنوان: | CRISPR-Cas12a exhibits metal-dependent specificity switching. |
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| المؤلفون: | Nguyen GT; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA., Schelling MA; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA., Raju A; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA., Buscher KA; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA., Sritharan A; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA., Sashital DG; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA. |
| المصدر: | Nucleic acids research [Nucleic Acids Res] 2024 Jul 18. Date of Electronic Publication: 2024 Jul 18. |
| Publication Model: | Ahead of Print |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Oxford University Press Country of Publication: England NLM ID: 0411011 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1362-4962 (Electronic) Linking ISSN: 03051048 NLM ISO Abbreviation: Nucleic Acids Res Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: 1992- : Oxford : Oxford University Press Original Publication: London, Information Retrieval ltd. |
| مستخلص: | Cas12a is the immune effector of type V-A CRISPR-Cas systems and has been co-opted for genome editing and other biotechnology tools. The specificity of Cas12a has been the subject of extensive investigation both in vitro and in genome editing experiments. However, in vitro studies have often been performed at high magnesium ion concentrations that are inconsistent with the free Mg2+ concentrations that would be present in cells. By profiling the specificity of Cas12a orthologs at a range of Mg2+ concentrations, we find that Cas12a switches its specificity depending on metal ion concentration. Lowering Mg2+ concentration decreases cleavage defects caused by seed mismatches, while increasing the defects caused by PAM-distal mismatches. We show that Cas12a can bind seed mutant targets more rapidly at low Mg2+ concentrations, resulting in faster cleavage. In contrast, PAM-distal mismatches cause substantial defects in cleavage following formation of the Cas12a-target complex at low Mg2+ concentrations. We observe differences in Cas12a specificity switching between three orthologs that results in variations in the routes of phage escape from Cas12a-mediated immunity. Overall, our results reveal the importance of physiological metal ion conditions on the specificity of Cas effectors that are used in different cellular environments. (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.) |
| التعليقات: | Update of: bioRxiv. 2024 Jan 17:2023.11.29.569287. doi: 10.1101/2023.11.29.569287. (PMID: 38076861) |
| معلومات مُعتمدة: | 1652661 National Science Foundation; GM140876 United States NH NIH HHS; Iowa State University |
| فهرسة مساهمة: | Local Abstract: [plain-language-summary] CRISPR-Cas systems are commonly used for biotechnology. Their specificity has been studied extensively and has previously been thought to be well understood. In this work, we asked a simple question about the effect of metal ion concentration on CRISPR specificity; the results are surprising and striking. At the actual metal ion concentrations found in cells, Cas12a specificity is inverted in comparison to the higher metal ion conditions that are typically used in test-tube assays. The specificity observed at lower metal ion concentration is more relevant under cellular conditions. |
| تواريخ الأحداث: | Date Created: 20240717 Latest Revision: 20240725 |
| رمز التحديث: | 20240726 |
| DOI: | 10.1093/nar/gkae613 |
| PMID: | 39019776 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 1362-4962 |
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| DOI: | 10.1093/nar/gkae613 |