Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology

Streptococcussuis is animportantzoonotic pathogen that causes severe invasive disease in pigs andhumans. Current methods for genome engineering of S.suis rely on the insertion of antibiotic resistancemarkers, which is time-consuming and labor-intensive and does notallow the precise introduction of small genomic mutations. Here wedeveloped a system for CRISPR-based genome editing in S. suis, utilizing linear DNA fragments for homologousrecombination (HR) and a plasmid-based negative selection system forbacteria not edited by HR. To enable the use of this system in otherbacteria, we engineered a broad-host-range replicon in the CRISPRplasmid. We demonstrated the utility of this system to rapidly introducemultiple gene deletions in successive rounds of genome editing andto make precise nucleotide changes in essential genes. Furthermore,we characterized a mechanism by which S. suis can escape killing by a targeted Cas9-sgRNA complex in the absenceof HR. A characteristic of this new mechanism is the presence of veryslow-growing colonies in a persister-like state that may allow forDNA repair or the introduction of mutations, alleviating Cas9 pressure.This does not impact the utility of CRISPR-based genome editing becausethe escape colonies are easily distinguished from genetically editedclones due to their small colony size. Our CRISPR-based editing systemis a valuable addition to the genetic toolbox for engineering of S. suis, as it accelerates the process of mutantconstruction and simplifies the removal of antibiotic markers betweensuccessive rounds of genome editing.