Functional characterization of a type I-F1 CRISPR-cas system from the clinical isolate Shewanella xiamenensis Sh95 reveals constitutive activity and plasmid-curing capability.

CRISPR-Cas are prokaryotic adaptive immunity defense systems that provide protection through RNA-guided recruited nucleases effectors.Despite the vast diversity in which CRISPR-Cas systems naturally exist, the single effectors from Class 2 were the most studied as genetic engineering tools. Later, the endogenous reprogramming of type I CRISPR-Cas systems showed promising results in diverse bacteria. However, the features and functions of the subtype I-F1 from Shewanella spp. members remain poorly characterized. Here, we report the analysis of the genetic context and activity of a type I-F1 CRISPR-Cas system within the species from the clinical isolate Shewanella xiamenensis Sh95 (SxCRISPR-Cas3). We show that this system transcribes constitutively and contains an internal promoter within cas3f open reading frame. Using endogenous reprogramming assays with synthetic mini CRISPR arrays, we demonstrate that the system is functionally active producing target plasmid interference and plasmid curing. Together, these results represent a simple methodology for obtaining colonies of S. xiamenensis Sh95 cured from the target plasmid and lay the foundation to the future exploration of SxCRISPR-Cas3 as a programmable interference tool.