Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering

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Title: Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering
Authors: Esquerra, Belén | Baquedano, Ignacio | Ruiz, Raul | Fernandez, Almudena | Montoliu, Lluis | Mojica, Francisco J.M.
Research Group/s: Microbiología Molecular
Center, Department or Service: Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología
Keywords: Cas9 | CRISPR | Genome editing | Metagenome | Recombineering
Issue Date: 25-Apr-2023
Publisher: Applied Microbiology International | John Wiley & Sons
Citation: Microbial Biotechnology. 2023, 16(7): 1505-1523.
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.
Sponsor: Conselleria de Innovación, Universidades, Ciencia y Sociedad Digital, Generalitat Valenciana (CIUCSD), Grant/Award Numbers: PROMETEO/2017/129 and PROMETEO/2021/057. Ministerio de Ciencia e Innovación (MICINN), Grant/Award Numbers: RTI2018-101223-B-I00 and PID2021-127644OB-I00.
ISSN: 1751-7915
DOI: 10.1111/1751-7915.14266
Language: eng
Type: info:eu-repo/semantics/article
Rights: © 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Peer Review: si
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Appears in Collections:INV - Microbiología Molecular - Artículos de Revistas

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