Sibr-Cas Enables Host-Independent and Universal CRISPR Genome Engineering in Prokaryotes

Homologous recombination combined with CRISPR-Cas counterselection is a powerful approach for genome editing in bacterial species. However, genome editing efficacy is dependent on host factors and (the efficiency of) DNA repair pathways of the host. To mitigate these constrains, we developed SIBR-Cas (Self-splicing Intron Based Riboswitch-Cas), which functions as a modular, tightly regulated and inducible system that can control the expression of FnCas12a in a wide range of bacterial species. We chose the Group I T4 bacteriophage thymidylate synthase (td) intron as the appropriate mechanism to control FnCas12a and repurposed it into a universal and inducible genome editing system. SIBR-Cas was applied successfully to knock-out several genes in three distinct wild type bacterial species (Escherichia coli, Pseudomonas putida and Flavobacterium IR1) with poor homologous recombination systems, without the use of exogenous recombinases nor use of inducible promoters. Compared to other genome engineering tools, SIBR-Cas is simple (one plasmid and independent of host factors), tight (little to no leakage), inducible (only active when inducer is applied in the medium) and widely applicable (can be applied to virtually any gene/RNA of interest and any bacterium under any promoter), allowing for the full utilization of non-model bacteria. Finally, we propose that SIBR-X (X stands for any gene/RNA of interest) can have a wider application as a universal gene expression control mechanism for virtually any gene of interest in prokaryotic organisms.