The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET
Summary: Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy trans...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2018-01-01
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| Series: | Cell Reports |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2211124717318727 |
| _version_ | 1818358385149476864 |
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| author | Mengyi Yang Sijia Peng Ruirui Sun Jingdi Lin Nan Wang Chunlai Chen |
| author_facet | Mengyi Yang Sijia Peng Ruirui Sun Jingdi Lin Nan Wang Chunlai Chen |
| author_sort | Mengyi Yang |
| collection | DOAJ |
| description | Summary: Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. : Yang et al. revealed significant conformational dynamics of Cas9 at global and local scales using single-molecule FRET. They uncovered surprising long-range allosteric communication between the HNH nuclease domain and the RNA/DNA heteroduplex at the PAM-distal end that serves as a proofreading checkpoint to govern the nuclease activity and specificity of Cas9. Keywords: CRISPR, Cas9, single-molecule, FRET, conformational dynamics, proofreading, off-target, allosteric communication, genome editing |
| first_indexed | 2024-12-13T20:28:09Z |
| format | Article |
| id | doaj.art-01bd962af0204cb282a41f354f4d7148 |
| institution | Directory Open Access Journal |
| issn | 2211-1247 |
| language | English |
| last_indexed | 2024-12-13T20:28:09Z |
| publishDate | 2018-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Reports |
| spelling | doaj.art-01bd962af0204cb282a41f354f4d71482022-12-21T23:32:30ZengElsevierCell Reports2211-12472018-01-01222372382The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRETMengyi Yang0Sijia Peng1Ruirui Sun2Jingdi Lin3Nan Wang4Chunlai Chen5School of Life Sciences, Tsinghua University, Beijing, 100084, China; Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, 100084, China; Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, 100084, ChinaSchool of Life Sciences, Tsinghua University, Beijing, 100084, China; Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, 100084, China; Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, 100084, ChinaSchool of Life Sciences, Tsinghua University, Beijing, 100084, China; Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, 100084, China; Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, 100084, ChinaSchool of Life Sciences, Tsinghua University, Beijing, 100084, China; Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, 100084, China; Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, 100084, ChinaSchool of Life Sciences, Tsinghua University, Beijing, 100084, China; Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, 100084, China; Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, 100084, ChinaSchool of Life Sciences, Tsinghua University, Beijing, 100084, China; Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, 100084, China; Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, 100084, China; Corresponding authorSummary: Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. : Yang et al. revealed significant conformational dynamics of Cas9 at global and local scales using single-molecule FRET. They uncovered surprising long-range allosteric communication between the HNH nuclease domain and the RNA/DNA heteroduplex at the PAM-distal end that serves as a proofreading checkpoint to govern the nuclease activity and specificity of Cas9. Keywords: CRISPR, Cas9, single-molecule, FRET, conformational dynamics, proofreading, off-target, allosteric communication, genome editinghttp://www.sciencedirect.com/science/article/pii/S2211124717318727 |
| spellingShingle | Mengyi Yang Sijia Peng Ruirui Sun Jingdi Lin Nan Wang Chunlai Chen The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET Cell Reports |
| title | The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET |
| title_full | The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET |
| title_fullStr | The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET |
| title_full_unstemmed | The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET |
| title_short | The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET |
| title_sort | conformational dynamics of cas9 governing dna cleavage are revealed by single molecule fret |
| url | http://www.sciencedirect.com/science/article/pii/S2211124717318727 |
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