Enhanced proofreading governs CRISPR–Cas9 targeting accuracy
| Autor: | Samuel H. Sternberg, Benjamin P. Kleinstiver, Ahmet Yildiz, J. Keith Joung, Jennifer A. Doudna, Janice S. Chen, Lucas B. Harrington, Yavuz S. Dagdas, Alexander A. Sousa, Moira M. Welch |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Streptococcus pyogenes General Science & Technology CRISPR-Associated Proteins Protein domain Biophysics 02 engineering and technology Computational biology Biology Cleavage (embryo) 01 natural sciences Article Substrate Specificity 03 medical and health sciences 0302 clinical medicine Protein Domains Genome editing MD Multidisciplinary Humans CRISPR Gene Editing Genetics Nuclease Multidisciplinary 010405 organic chemistry Chemistry Cas9 Genetic Variation Endonucleases 021001 nanoscience & nanotechnology 0104 chemical sciences Enzyme Activation 030104 developmental biology Förster resonance energy transfer Mutagenesis biology.protein Proofreading CRISPR-Cas Systems 0210 nano-technology 030217 neurology & neurosurgery Biotechnology |
| Zdroj: | Nature, vol 550, iss 7676 Nature |
| ISSN: | 1476-4687 0028-0836 |
| DOI: | 10.1038/nature24268 |
| Popis: | The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown. Using single-molecule Forster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target mismatches and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we identified residues within REC3 involved in mismatch sensing and designed a new hyper-accurate Cas9 variant (HypaCas9) that retains robust on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing. |
| Databáze: | OpenAIRE |
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