Streamlined CRISPR genome engineering in wild-type bacteria using SIBR-Cas

Abstract CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and widely applicable genome engin...

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Bibliographic Details
Published in:Nucleic acids research 2021-11, Vol.49 (19), p.11392-11404
Main Authors: Patinios, Constantinos, Creutzburg, Sjoerd C A, Arifah, Adini Q, Adiego-Pérez, Belén, Gyimah, Evans A, Ingham, Colin J, Kengen, Servé W M, van der Oost, John, Staals, Raymond H J
Format: Article
Language:English
Subjects:
Base Pairing
Base Sequence
CRISPR-Cas Systems
Escherichia coli - genetics
Escherichia coli - metabolism
Flavobacterium - genetics
Flavobacterium - metabolism
Gene Editing - methods
Gene Knockout Techniques - methods
Genome, Bacterial
Homologous Recombination
Introns
Nucleic Acid Conformation
Pseudomonas putida - genetics
Pseudomonas putida - metabolism
Riboswitch
RNA Splicing
RNA, Bacterial - genetics
RNA, Bacterial - metabolism
Synthetic Biology and Bioengineering
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Summary:Abstract CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and widely applicable genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for tight and inducible control over CRISPR-Cas counter-selection. This control delays CRISPR-Cas counter-selection, granting more time for the editing event (e.g. by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three wild-type bacteria species (Escherichia coli MG1655, Pseudomonas putida KT2440 and Flavobacterium IR1) with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a simple gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkab893