Biology subjects

Brouns, S.

Publications and source records attributed to Brouns, S..

3 recordsLinked to original sources

bioRxiv2019

Selective Prespacer Processing Ensures Precise CRISPR-Cas Adaptation

CRISPR-Cas immunity protects prokaryotes against foreign genetic elements. CRISPR-Cas uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers). It remains elusive how Cas1-Cas2 acquires spacers from cellular DNA fragments (prespacers) and how it integrates them into the CRISPR array in the correct orientation. By using the high spatiotemporal resolution of single-molecule fluorescence, we reveal that Cas1-Cas2 obtains prespacers in various forms including single-stranded DNA and partial duplexes by selecting them in the DNA-length and PAM-dependent manner. Furthermore, we identify DnaQ exonucleases as enzymes that can mature the Cas1-Cas2-loaded precursor prespacers into an integration-competent size. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and subsequent spacer integration in the correct orientation. This kinetic coordination in prespacer selection and PAM trimming provides comprehensive understanding of the mechanisms that underlie the integration of functional spacers in the CRISPR array.

biophysics↗

bioRxiv2019

Direct visualization of native CRISPR target search in live bacteria reveals Cascade DNA surveillance mechanism

CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here we visualized individual Cascade complexes in a native type I CRISPR-Cas system. We uncovered an exponential relationship between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA ([~]30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and impact CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.\n\nOne sentence summaryThe results from in vivo tracking of single CRISPR RNA-surveillance complexes in the native host cell explain their ability to rapidly recognize invader sequences.

microbiology↗

bioRxiv2019

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage

Microbiomes are vast communities of microbes and viruses that populate all natural ecosystems. Viruses have been considered the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared to other environments. Here we investigate the origin, evolution, and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboratory, we obtained DNA sequences of crAssphage from over one-third of the worlds countries, and showed that its phylogeography is locally clustered within countries, cities, and individuals. We also found colinear crAssphage-like genomes in both Old-World and New-World primates, challenging genomic mosaicism and suggesting that the association of crAssphage with primates may be millions of years old. We conclude that crAssphage is a benign globetrotter virus that may have co-evolved with the human lineage and an integral part of the normal human gut virome.

microbiology↗