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Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering |
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| Authors: | Belen Esquerra-Ruvira Ignacio Baquedano Raul Ruiz Almudena Fernandez Lluis Montoliu Francisco J M Mojica |
| Institution: | 1. Department of Physiology, Genetics and Microbiology, University of Alicante, Alicante, Spain
Contribution: Conceptualization (equal), Data curation (equal), Formal analysis (equal), Investigation (equal), Methodology (equal), Resources (equal), Software (equal), Validation (equal), Visualization (equal), Writing - original draft (supporting), Writing - review & editing (supporting);2. Department of Physiology, Genetics and Microbiology, University of Alicante, Alicante, Spain Contribution: Conceptualization (equal), Data curation (equal), Formal analysis (equal), Investigation (equal), Methodology (equal), Resources (equal), Software (equal), Validation (equal), Visualization (equal), Writing - original draft (equal), Writing - review & editing (equal);3. Department of Physiology, Genetics and Microbiology, University of Alicante, Alicante, Spain;4. Department of Molecular and Cellular Biology, National Centre for Biotechnology (CNB-CSIC), Madrid, Spain Centre for Biomedical Network Research on Rare Diseases (CIBERER-ISCIII), Madrid, Spain Contribution: Conceptualization (equal), Investigation (equal), Methodology (equal), Resources (equal), Supervision (equal), Validation (equal), Writing - review & editing (supporting);5. Department of Molecular and Cellular Biology, National Centre for Biotechnology (CNB-CSIC), Madrid, Spain |
| Abstract: | Non-coding RNAs (crRNAs) produced from clustered regularly interspaced short palindromic repeats (CRISPR) loci and CRISPR-associated (Cas) proteins of the prokaryotic CRISPR-Cas systems form complexes that interfere with the spread of transmissible genetic elements through Cas-catalysed cleavage of foreign genetic material matching the guide crRNA sequences. The easily programmable targeting of nucleic acids enabled by these ribonucleoproteins has facilitated the implementation of CRISPR-based molecular biology tools for in vivo and in vitro modification of DNA and RNA targets. Despite the diversity of DNA-targeting Cas nucleases so far identified, native and engineered derivatives of the Streptococcus pyogenes SpCas9 are the most widely used for genome engineering, at least in part due to their catalytic robustness and the requirement of an exceptionally short motif (5′-NGG-3′ PAM) flanking the target sequence. However, the large size of the SpCas9 variants impairs the delivery of the tool to eukaryotic cells and smaller alternatives are desirable. Here, we identify in a metagenome a new CRISPR-Cas9 system associated with a smaller Cas9 protein (EHCas9) that targets DNA sequences flanked by 5′-NGG-3′ PAMs. We develop a simplified EHCas9 tool that specifically cleaves DNA targets and is functional for genome editing applications in prokaryotes and eukaryotic cells. |
| Keywords: | Cas9 CRISPR genome editing metagenome recombineering |