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.

Virus infection modulates male sexual behaviour in Caenorhabditis elegans.

Mating dynamics follow from natural selection on mate choice and individuals maximizing their reproductive success. Mate discrimination reveals itself by a plethora of behaviours and morphological characteristics, each of which can be affected by pathogens. A key question is how pathogens affect mate choice and outcrossing behaviour. Here we investigated the effect of Orsay virus on the mating dynamics of the androdiecious (male and hermaphrodite) nematode Caenorhabditis elegans. We tested genetically distinct strains and found that viral susceptibility differed between sexes in a genotype-dependent manner with males of reference strain N2 being more resistant than hermaphrodites. Males displayed a constitutively higher expression of intracellular pathogen response (IPR) genes, whereas the antiviral RNAi response did not have increased activity in males. Subsequent monitoring of sex ratios over 10 generations revealed that viral presence can change mating dynamics in isogenic populations. Sexual attraction assays showed that males preferred mating with uninfected rather than infected hermaphrodites. Together our results illustrate for the first time that viral infection can significantly affect male mating choice and suggest altered mating dynamics as a novel cause benefitting outcrossing under pathogenic stress conditions in C. elegans.