A Minimal CRISPR-Cas3 System for Rapid Large-Scale Genome Manipulation

CRISPR-Cas systems are a diverse branch of prokaryotic immune systems that have been co-opted for prokaryotic and eukaryotic gene editing. Here, we present CRISPR-Cas3 based editing, an easily-programmable genome engineering technique that allows for the rapid generation of large genomic deletions. Cas3, the signature enzyme of CRISPR-Cas Type I systems, possesses coupled nuclease and helicase activity, allowing for processive degradation of a targeted region. While the leading CRISPR-Cas tools (e.g. Cas9 and Cas12a) allow for genetic edits like small insertions and deletions, these enzymes are limited when the desired manipulation scales in size (>10 kb). With this in mind, we optimized a CRISPR-Cas3 system to generate both random and pre-designed large deletions (up to 250 kb) in Pseudomonas aeruginosa. Iterative transformation of one strain with ten distinct guides yielded ten deletions resulting in a reduction of 10% of the genome in thirty days. Additionally, using one guide to target loci that exist in multiple copies within a single chromosome allowed us to simultaneously delete more than one portion of the genome. To test the portability of CRISPR-Cas3, we induced large deletions in both Pseudomonas syringae and Escherichia coli using an “all-in-one” vector expressing cas genes and a guide. Deletion efficiencies in these orthogonal systems remained high. Thus, a tool like CRISPR-Cas3 will facilitate rapid strain manipulation for synthetic biological and metabolic engineering purposes as well as the analysis of regions of unknown function in diverse organisms for basic biological research.