A plasmid toolset for CRISPR-mediated genome editing and CRISPRi gene regulation in Escherichia coli.

CRISPR technologies have become standard laboratory tools for genetic manipulations across all kingdoms of life. Despite their origins in bacteria, the development of CRISPR tools for engineering bacteria has been slower than for eukaryotes; nevertheless, their function and application for genome engineering and gene regulation via CRISPR interference (CRISPRi) has been demonstrated in various bacteria, and adoption has become more widespread. Here, we provide simple plasmid-based systems for genome editing (gene knockouts/knock-ins, and genome integration of large DNA fragments) and CRISPRi in E. coli using a CRISPR-Cas12a system. The described genome engineering protocols allow markerless deletion or genome integration in just seven working days with high efficiency (> 80% and 50%, respectively), and the CRISPRi protocols allow robust transcriptional repression of target genes (> 90%) with a single cloning step. The presented minimized plasmids and their associated design and experimental protocols provide efficient and effective CRISPR-Cas12 genome editing, genome integration and CRISPRi implementation. These simple-to-use systems and protocols will allow the easy adoption of CRISPR technology by any laboratory.