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 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.

Implementation of the β-hydroxyaspartate cycle increases growth performance of Pseudomonas putida on the PET monomer ethylene glycol

“…Further adaptations required mutations of the regulatory network of P. putida KT2440, in particular, lldR , regB and a LysR-family regulator gene of unknown function, which showed pleiotropic effects on the metabolic network of P. putida KT2440. This multitude of changes to fully embed the BHAC with the genetic and metabolic network of the cell could most likely not have been realized by rational engineering, even though there is a broad variety of advanced genetic tools for P. putida (Martínez-García et al, 2017; Choi et al, 2020; Wirth et al, 2020; Patinios et al, 2021; Velázquez et al, 2021; Abdullah et al, 2022; Liu et al, 2022). In our view, the different changes in production of enzymes and respiratory complexes would have been nearly impossible to achieve by overexpressing genes or introducing point mutations.…”

Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria

“…The copyright holder for this preprint this version posted September 14, 2022. ; https://doi.org/10.1101/2022.09.14.507927 doi: bioRxiv preprint 10 they still yielded higher colony counts than the original gRNA (e.g., 13-fold for atgRNA1 and 20fold for atgRNA2 for gfp replacement with 750-bp homology arms). The atgRNAs also always yielded higher editing efficiencies than the original gRNA, with longer homology arms consistently increasing the editing efficiency.…”

Metabolic engineering of Clostridium autoethanogenum for ethyl acetate production from CO

“…The pta gene (CAETHG_3358) was targeted using SIBR-Cas gene editing techniques as described previously [25]. The targeting vector was based on the backbone pMTL83151 with expression of the FnCas12a variant with Int3 [25] under control of the fdx promoter from Clostridium sporogenes [23], a crRNA under control of the araE promoter from Clostridium acetobutylicum [28] and homology arms of ca. 1 kb up-and downstream of pta.…”