Abstract
CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defence against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not interfere equally with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. We propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory-acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses.This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.
Highlights
Phages often vastly outnumber their bacterial hosts in a variety of environments [1]
clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are composed of a CRISPR array—a series of ‘spacers’ of foreign sequence alternating with repeats that are transcribed into CRISPR RNAs—and cas genes
In our spacer acquisition experiment, we identified a subset of spacers that target a mu-like region in the V. cholerae large chromosome, suggesting that CRISPR targeting of the mu-like region was advantageous in overcoming phage inducible chromosomal island-like elements (PLEs)
Summary
Phages often vastly outnumber their bacterial hosts in a variety of environments [1]. Bacteria have evolved numerous mechanisms for phage defence, including adaptive immunity via clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins [2,3]. Together with crRNAs, Cas proteins defend against foreign nucleic acids, such as the genome of an infecting phage, through a three-step process: adaptation, crRNA and cas gene expression, and interference. A foreign DNA fragment is incorporated into the CRISPR array to provide a molecular memory of the challenges that the host cell has faced. This CRISPR array is expressed and processed into individual crRNAs, which complex with Cas proteins and survey the cell for.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
