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CRISPR-Cas Effector Specificity and Cleavage Site Determine Phage Escape Outcomes

Michael A. Schelling, Giang T. Nguyen, Dipali G. Sashital


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 protospacer adjacent motif (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 preexisting mismatches against the genomic targets in phage DNA. We find that most preexisting crRNA mismatches lead to phage escape, 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. 


CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems are adaptive immune systems found in bacteria and archaea [1–3]. These systems use ribonucleoprotein effector complexes to find and destroy foreign nucleic acids that have entered the cell. 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. Bacteria can acquire new spacer sequences that allow them to mount an immune response against threats they have not previously encountered.

Materials and method    

Expression plasmid construction

All primers and plasmids used in this study are listed in S1 Table. Cas12a and Cas9 expression plasmids were constructed using pACYCDuet-1. A gene expressing FnCas12a or SpCas9 was inserted downstream of a pBAD promoter in pACYCDuet-1 using Gibson assembly. Promoters were replaced with a previously described low copy P4 promoter [49] using 1 piece Gibson assembly. crRNA expression plasmids were constructed using pUC19. A pBAD promoter was inserted into pUC19 in the multiple cloning site with Gibson assembly. “Round the horn” PCR and ligation was used to add a mini CRISPR array with 1 or 2 spacers downstream of the pBAD promoter. The same method was used to replace mini CRISPR arrays with Cas9 sgRNA expression constructs.


crRNA mismatches throughout the spacer decrease phage protection provided by Cas12a

To investigate the effect of crRNA mismatches on phage immunity provided by Cas12a, we developed a heterologous type V-A CRISPR-Cas12a system in Escherichia coli. We expressed Cas12a from Francisella novicida and various pre-crRNAs from 2 different plasmids in E. coli K12 using a strong inducible promoter (PBAD) or a relatively weak constitutive promoter [49,63]. We infected these cells with lambda phage to measure the immunity provided by Cas12a (Fig 1A). We used λvir, a mutant of lambda phage that cannot engage in lysogeny and is locked into the lytic mode of replication. This eliminates CRISPR self-targeting that could occur if a target phage becomes a lysogen in the bacterial genome. We chose 2 lambda genomic targets: one target was in an intergenic region upstream of gene J and the other target was inside the coding region of gene L (Fig 1A). Both genes encode essential structural tail tip proteins. The Cas12a expression system exhibited a high level of protection for both promoters, with targeting crRNAs showing about 106 fold less phage infection than the non-targeting control (Fig 1B).


In order for Cas12a to be an effective immune effector, it must provide immunity from bacteriophage in diverse conditions. The physical environment controls which bacteria are exposed to which phages and the dispersal of new phage particles after infection and lysis [9,68]. We found that Cas12a overall provided more robust immunity on solid media than in liquid culture. The effect of seed crRNA mismatches using either media correlated with the deleterious effect of the crRNA mismatch on the rate of cleavage in vitro. Mid-target and PAM-distal mismatches, however, showed a much more drastic effect in liquid culture than defects observed in vitro or on solid media when Cas12a was expressed from a strong promoter, causing eventual lysis of the bacterial population, sometimes at a rate similar to seed mismatches. These results strongly indicate that the effect of crRNA mismatches varies depending on the environment where phage exposure may occur.

Citation: Schelling MA, Nguyen GT, Sashital DG (2023) CRISPR-Cas effector specificity and cleavage site determine phage escape outcomes. PLoS Biol 21(4): e3002065.

Academic Editor: Jeremy J. Barr, Monash University, AUSTRALIA

Received: July 11, 2022; Accepted: March 6, 2023; Published: April 14, 2023

Copyright: © 2023 Schelling et al. 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.

Data Availability: All relevant data are within the paper and its Supporting Information files.” Scripts used to analyze high-throughput sequencing data are available at

Funding: Financial support for this research was provided by National Science Foundation award 1652661 (to D.G.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors declare no competing interests.

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