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

Streptococcussuis is an
important
zoonotic pathogen that causes severe invasive disease in pigs and
humans. Current methods for genome engineering of S.
suis rely on the insertion of antibiotic resistance
markers, which is time-consuming and labor-intensive and does not
allow the precise introduction of small genomic mutations. Here we
developed a system for CRISPR-based genome editing in S. suis, utilizing linear DNA fragments for homologous
recombination (HR) and a plasmid-based negative selection system for
bacteria not edited by HR. To enable the use of this system in other
bacteria, we engineered a broad-host-range replicon in the CRISPR
plasmid. We demonstrated the utility of this system to rapidly introduce
multiple gene deletions in successive rounds of genome editing and
to 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 absence
of HR. A characteristic of this new mechanism is the presence of very
slow-growing colonies in a persister-like state that may allow for
DNA repair or the introduction of mutations, alleviating Cas9 pressure.
This does not impact the utility of CRISPR-based genome editing because
the escape colonies are easily distinguished from genetically edited
clones due to their small colony size. Our CRISPR-based editing system
is a valuable addition to the genetic toolbox for engineering of S. suis, as it accelerates the process of mutant
construction and simplifies the removal of antibiotic markers between
successive rounds of genome editing.