![]() Īn important function of CRISPR-Cas systems is to prevent infection by bacteriophages, which can have significant impact on the composition of a bacterial population. Bacteria can acquire new spacer sequences that allow them to mount an immune response against threats they have not previously encountered. CRISPR effector complexes are guided by a CRISPR RNA (crRNA) to a nucleic acid target that is complementary to a section of the crRNA called the spacer. ![]() These systems use ribonucleoprotein effector complexes to find and destroy foreign nucleic acids that have entered the cell. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors declare no competing interests.ĬRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems are adaptive immune systems found in bacteria and archaea. įunding: Financial support for this research was provided by National Science Foundation award 1652661 (to D.G.S.). This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the paper and its Supporting Information files.” Scripts used to analyze high-throughput sequencing data are available at. Received: JAccepted: MaPublished: April 14, 2023Ĭopyright: © 2023 Schelling et al. PLoS Biol 21(4):Īcademic Editor: Jeremy J. These results demonstrate that Cas effector mismatch tolerance, existing target mismatches, and cleavage site strongly influence phage evolution.Ĭitation: Schelling MA, Nguyen GT, Sashital DG (2023) CRISPR-Cas effector specificity and cleavage site determine phage escape outcomes. Expression of multiple mismatched crRNAs prevented new mutations from arising in multiple targeted locations, allowing Cas12a mismatch tolerance to provide stronger and longer-term protection. However, similar experiments with Cas9 did not result in emergence of PAM-distal mismatches, suggesting that cut-site location and subsequent DNA repair may influence the location of escape mutations within target regions. In vitro cleavage and phage competition assays show that dual PAM-distal mismatches are significantly more deleterious than combinations of seed and PAM-distal mismatches, resulting in this selection. Unexpectedly, our results reveal that a preexisting mismatch in the PAM-distal region results in selection of mutations in the PAM-distal region of the target. Mismatches at all locations in the target accelerated emergence of mutant phage, including mismatches that greatly slowed cleavage in vitro. We used high-throughput sequencing to examine the target regions of phage genomes following CRISPR challenge. We find that most preexisting crRNA mismatches lead to phage escape, regardless of whether the mismatches ablate Cas12a cleavage in vitro. Here, we tested defense against lambda phage provided by Cas12a-crRNAs containing preexisting mismatches against the genomic targets in phage DNA. The effect of this mismatch tolerance has not been extensively studied in the context of phage defense. ![]() However, previous specificity studies of Cas effectors, including the class 2 endonuclease Cas12a, have revealed a high degree of tolerance of single mismatches. Phages escape CRISPR-based immunity mainly through mutations in the protospacer adjacent motif (PAM) and seed regions. CRISPR-mediated interference relies on complementarity between a guiding CRISPR RNA (crRNA) and target nucleic acids to provide defense against bacteriophage.
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