期刊
NUCLEIC ACIDS RESEARCH
卷 49, 期 19, 页码 11392-11404出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkab893
关键词
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资金
- Graduate School VLAG, Wageningen University and Research, Netherlands
- VENI grant from 'The Netherlands Organization for Scientific Research' (NWO) [016.Veni.171.047]
- 'European Research Council' (ERC) [ERC-AdG-834279]
- ERC Advanced Grant (European Research Council) [ERC-AdG-834279]
A simple and widely applicable genome engineering tool SIBR-Cas was developed in this study, utilizing a theophylline-dependent self-splicing intron to control CRISPR-Cas counter-selection and delay editing events. This method was successfully applied to knock-out genes in multiple bacteria species with poor homologous recombination systems, making it suitable for most bacteria. Additionally, SIBR is proposed to have a broader application as a gene expression and gene regulation control mechanism in bacteria.
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.
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