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Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations

Base editors are recent multiplex gene editing tools derived from the Cas9 nuclease of Streptomyces pyogenes. They can target and modify a single nucleotide in the genome without inducing double-strand breaks (DSB) of the DNA helix. As such, they hold great potential for the engineering of microbes...

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Autores principales: Seys, François M., Humphreys, Christopher M., Tomi-Andrino, Claudio, Li, Qi, Millat, Thomas, Yang, Sheng, Minton, Nigel P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10366002/
https://www.ncbi.nlm.nih.gov/pubmed/37496853
http://dx.doi.org/10.3389/fbioe.2023.1211197
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author Seys, François M.
Humphreys, Christopher M.
Tomi-Andrino, Claudio
Li, Qi
Millat, Thomas
Yang, Sheng
Minton, Nigel P.
author_facet Seys, François M.
Humphreys, Christopher M.
Tomi-Andrino, Claudio
Li, Qi
Millat, Thomas
Yang, Sheng
Minton, Nigel P.
author_sort Seys, François M.
collection PubMed
description Base editors are recent multiplex gene editing tools derived from the Cas9 nuclease of Streptomyces pyogenes. They can target and modify a single nucleotide in the genome without inducing double-strand breaks (DSB) of the DNA helix. As such, they hold great potential for the engineering of microbes that lack effective DSB repair pathways such as homologous recombination (HR) or non-homologous end-joining (NHEJ). However, few applications of base editors have been reported in prokaryotes to date, and their advantages and drawbacks have not been systematically reported. Here, we used the base editors Target-AID and Target-AID-NG to introduce nonsense mutations into four different coding sequences of the industrially relevant Gram-positive bacterium Clostridium autoethanogenum. While up to two loci could be edited simultaneously using a variety of multiplexing strategies, most colonies exhibited mixed genotypes and most available protospacers led to undesired mutations within the targeted editing window. Additionally, fifteen off-target mutations were detected by sequencing the genome of the resulting strain, among them seven single-nucleotide polymorphisms (SNP) in or near loci bearing some similarity with the targeted protospacers, one 15 nt duplication, and one 12 kb deletion which removed uracil DNA glycosylase (UDG), a key DNA repair enzyme thought to be an obstacle to base editing mutagenesis. A strategy to process prokaryotic single-guide RNA arrays by exploiting tRNA maturation mechanisms is also illustrated.
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spelling pubmed-103660022023-07-26 Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations Seys, François M. Humphreys, Christopher M. Tomi-Andrino, Claudio Li, Qi Millat, Thomas Yang, Sheng Minton, Nigel P. Front Bioeng Biotechnol Bioengineering and Biotechnology Base editors are recent multiplex gene editing tools derived from the Cas9 nuclease of Streptomyces pyogenes. They can target and modify a single nucleotide in the genome without inducing double-strand breaks (DSB) of the DNA helix. As such, they hold great potential for the engineering of microbes that lack effective DSB repair pathways such as homologous recombination (HR) or non-homologous end-joining (NHEJ). However, few applications of base editors have been reported in prokaryotes to date, and their advantages and drawbacks have not been systematically reported. Here, we used the base editors Target-AID and Target-AID-NG to introduce nonsense mutations into four different coding sequences of the industrially relevant Gram-positive bacterium Clostridium autoethanogenum. While up to two loci could be edited simultaneously using a variety of multiplexing strategies, most colonies exhibited mixed genotypes and most available protospacers led to undesired mutations within the targeted editing window. Additionally, fifteen off-target mutations were detected by sequencing the genome of the resulting strain, among them seven single-nucleotide polymorphisms (SNP) in or near loci bearing some similarity with the targeted protospacers, one 15 nt duplication, and one 12 kb deletion which removed uracil DNA glycosylase (UDG), a key DNA repair enzyme thought to be an obstacle to base editing mutagenesis. A strategy to process prokaryotic single-guide RNA arrays by exploiting tRNA maturation mechanisms is also illustrated. Frontiers Media S.A. 2023-07-10 /pmc/articles/PMC10366002/ /pubmed/37496853 http://dx.doi.org/10.3389/fbioe.2023.1211197 Text en Copyright © 2023 Seys, Humphreys, Tomi-Andrino, Li, Millat, Yang and Minton. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Seys, François M.
Humphreys, Christopher M.
Tomi-Andrino, Claudio
Li, Qi
Millat, Thomas
Yang, Sheng
Minton, Nigel P.
Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations
title Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations
title_full Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations
title_fullStr Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations
title_full_unstemmed Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations
title_short Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations
title_sort base editing enables duplex point mutagenesis in clostridium autoethanogenum at the price of numerous off-target mutations
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10366002/
https://www.ncbi.nlm.nih.gov/pubmed/37496853
http://dx.doi.org/10.3389/fbioe.2023.1211197
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