CRISPR-Cas effector specificity and target mismatches determine phage escape outcomes

CRISPR-mediated interference relies on complementarity between a guiding CRISPR RNA (crRNA) and target nucleic acids to provide defense against bacteriophage. Phages escape CRISPR-based immunity mainly through mutations in the PAM and seed regions. However, previous specificity studies of Cas effectors, including the class 2 endonuclease Cas12a, have revealed a high degree of tolerance of single mismatches. The effect of this mismatch tolerance has not been extensively studied in the context of phage defense. Here, we tested defense against lambda phage provided by Cas12a-crRNAs containing pre-existing mismatches against the genomic targets in phage DNA. We observe a correlation between Cas12a mismatch tolerance in vitro and phage defense on solid media. However, in liquid media, we find that most pre-existing crRNA mismatches lead to phage escape and lysis, regardless of whether the mismatches ablate Cas12a cleavage in vitro. We used high-throughput sequencing to examine the target regions of phage genomes following CRISPR challenge. Mismatches at all locations in the target accelerated emergence of mutant phage, including mismatches that greatly slowed cleavage in vitro. Mutations arose near the existing mismatch, in some cases resulting in multiple PAM-distal mismatches allowing for phage escape. Similar experiments with Cas9 showed the location of emergent target mutations was unaffected by pre-existing crRNA-target mismatches. 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. These results demonstrate that Cas effector mismatch tolerance and existing target mismatches strongly influence phage evolution.