| Institution: | 1. Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), c/ Nicolás Cabrera 1, Campus de Cantoblanco-UAM, Madrid, Spain;2. Department of Theoretical Condensed Matter Physics, Universidad Autónoma de Madrid, Spain;3. Department of Biotechnology, Universidad Francisco de Vitoria, Pozuelo de Alarcón, Madrid, Spain;1. State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Hongshan Laboratory, Hubei Key Laboratory of Industrial Biotechnology, School of life science, Hubei University, Wuhan 430062, Hubei, China;2. State Key Laboratory of Physical Chemistry of Solid Sur-faces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 360015, Fujian, China;3. Max-Planck-Institut für Kohlenforschung, 45470 Muelheim, Germany;4. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China;1. Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA;2. Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA;1. EMBL Hamburg Site, c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany;2. Magnetic Resonance Center CERM, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy;3. Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy;1. Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA;2. Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA;3. Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027, USA;4. Center for Molecular Microscopy, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA;5. Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA |
| Abstract: | The RNA-targeting type VI CRISPR-Cas effector complexes are widely used in biotechnology applications such as gene knockdown, RNA editing, and molecular diagnostics. Compared with Cas13a from mesophilic organisms, a newly discovered Cas13a from thermophilic bacteria Thermoclostridium caenicola (TccCas13a) shows low sequence similarity, high thermostability, and lacks pre-crRNA processing activity. The thermostability of TccCas13a has been harnessed to make a sensitive and robust tool for nucleic acid detection. Here we present the structures of TccCas13a-crRNA binary complex at 2.8 Å, and TccCas13a at 3.5 Å. Although TccCas13a shares a similarly bilobed architecture with other mesophilic organism-derived Cas13a proteins, TccCas13a displayed distinct structure features. Specifically, it holds a long crRNA 5′-flank, forming extensive polar contacts with Helical-1 and HEPN2 domains. The detailed analysis of the interaction between crRNA 5′-flank and TccCas13a suggested lack of suitable nucleophile to attack the 2′-OH of crRNA 5′-flank may explain why TccCas13a fails to cleave pre-crRNA. The stem-loop segment of crRNA spacer toggles between double-stranded and single-stranded conformational states, suggesting a potential safeguard mechanism for target recognition. Superimposition of the structures of TccCas13a and TccCas13a-crRNA revealed several conformational changes required for crRNA loading, including dramatic movement of Helical-2 domain. Collectively, these structural insights expand our understanding into type VI CRISPR-Cas effectors, and would facilitate the development of TccCas13a-based applications. |
