Bacterial resistance to CRISPR-Cas antimicrobials.

In the age of antibiotic resistance and precise microbiome engineering, CRISPR-Cas antimicrobials promise to have a substantial impact on the way we treat diseases in the future. However, the efficacy of these antimicrobials and their mechanisms of resistance remain to be elucidated. We systematically investigated how a target E. coli strain can escape killing by episomally-encoded CRISPR-Cas9 antimicrobials. Using Cas9 from Streptococcus pyogenes (SpCas9) we studied the killing efficiency and resistance mutation rate towards CRISPR-Cas9 antimicrobials and elucidated the underlying genetic alterations. We find that killing efficiency is not correlated with the number of cutting sites or the type of target. While the number of targets did not significantly affect efficiency of killing, it did reduce the emergence of chromosomal mutations conferring resistance. The most frequent target of resistance mutations was the plasmid-encoded SpCas9 that was inactivated by bacterial genome rearrangements involving translocation of mobile genetic elements such as insertion elements. This resistance mechanism can be overcome by re-introduction of an intact copy of SpCas9. The work presented here provides a guide to design strategies that reduce resistance and improve the activity of CRISPR-Cas antimicrobials.

Compatibility of Evolutionary Responses to Constituent Antibiotics Drive Resistance Evolution to Drug Pairs.

Antibiotic combinations are considered a relevant strategy to tackle the global antibiotic resistance crisis since they are believed to increase treatment efficacy and reduce resistance evolution (WHO treatment guidelines for drug-resistant tuberculosis: 2016 update.). However, studies of the evolution of bacterial resistance to combination therapy have focused on a limited number of drugs and have provided contradictory results (Lipsitch, Levin BR. 1997; Hegreness et al. 2008; Munck et al. 2014). To address this gap in our understanding, we performed a large-scale laboratory evolution experiment, adapting eight replicate lineages of Escherichia coli to a diverse set of 22 different antibiotics and 33 antibiotic pairs. We found that combination therapy significantly limits the evolution of de novode novo resistance in E. coli, yet different drug combinations vary substantially in their propensity to select for resistance. In contrast to current theories, the phenotypic features of drug pairs are weak predictors of resistance evolution. Instead, the resistance evolution is driven by the relationship between the evolutionary trajectories that lead to resistance to a drug combination and those that lead to resistance to the component drugs. Drug combinations requiring a novel genetic response from target bacteria compared with the individual component drugs significantly reduce resistance evolution. These data support combination therapy as a treatment option to decelerate resistance evolution and provide a novel framework for selecting optimized drug combinations based on bacterial evolutionary responses.

Chromosomal barcoding as a tool for multiplexed phenotypic characterization of laboratory evolved lineages.

Adaptive laboratory evolution is an important tool to evolve organisms to increased tolerance towards different physical and chemical stress. It is applied to study the evolution of antibiotic resistance as well as genetic mechanisms underlying improvements in production strains. Adaptive evolution experiments can be automated in a high-throughput fashion. However, the characterization of the resulting lineages can become a time consuming task, when the performance of each lineage is evaluated individually. Here, we present a novel method for the markerless insertion of randomized genetic barcodes into the genome of Escherichia coli using a novel dual-auxotrophic selection approach. The barcoded E. coli library allows multiplexed phenotyping of evolved strains in pooled competition experiments. We use the barcoded library in an adaptive evolution experiment; evolving resistance towards three common antibiotics. Comparing this multiplexed phenotyping with conventional susceptibility testing and growth-rate measurements we can show a significant positive correlation between the two approaches. Use of barcoded bacterial strain libraries for individual adaptive evolution experiments drastically reduces the workload of characterizing the resulting phenotypes and enables prioritization of lineages for in-depth characterization. In addition, barcoded clones open up new ways to profile community dynamics or to track lineages in vivo or situ.

Dependency of Heterochromatin Domains on Replication Factors.

Chromatin structure regulates both genome expression and dynamics in eukaryotes, where large heterochromatic regions are epigenetically silenced through the methylation of histone H3K9, histone deacetylation, and the assembly of repressive complexes. Previous genetic screens with the fission yeast Schizosaccharomyces pombe have led to the identification of key enzymatic activities and structural constituents of heterochromatin. We report here on additional factors discovered by screening a library of deletion mutants for silencing defects at the edge of a heterochromatic domain bound by its natural boundary-the IR-R+ element-or by ectopic boundaries. We found that several components of the DNA replication progression complex (RPC), including Mrc1/Claspin, Mcl1/Ctf4, Swi1/Timeless, Swi3/Tipin, and the FACT subunit Pob3, are essential for robust heterochromatic silencing, as are the ubiquitin ligase components Pof3 and Def1, which have been implicated in the removal of stalled DNA and RNA polymerases from chromatin. Moreover, the search identified the cohesin release factor Wpl1 and the forkhead protein Fkh2, both likely to function through genome organization, the Ssz1 chaperone, the Fkbp39 proline cis-trans isomerase, which acts on histone H3P30 and P38 in Saccharomyces cerevisiae, and the chromatin remodeler Fft3. In addition to their effects in the mating-type region, to varying extents, these factors take part in heterochromatic silencing in pericentromeric regions and telomeres, revealing for many a general effect in heterochromatin. This list of factors provides precious new clues with which to study the spatiotemporal organization and dynamics of heterochromatic regions in connection with DNA replication.