NEW YORK (GenomeWeb) – In two separate studies published in Science today, researchers from Jennifer Doudna's lab at the University of California, Berkeley, and a team led by the University of California, San Francisco's Joseph Bondy-Denomy are reporting the discovery of 15 total new CRISPR inhibitors.
Using a comprehensive bioinformatic and experimental screening approach, Doudna's team identified three different inhibitors that block or diminish CRISPR-Cas12a-mediated genome editing in human cells. While bacteria harboring Cas9 or Cas3 adaptive immune systems sometimes acquire genetic elements that encode Cas9, Cas3, or Cascade complex anti-CRISPR (Acr) proteins, this is the first instance in which inhibitors have been found for Cas12a, the researchers noted.
They also found a widespread connection between CRISPR self-targeting and the prevalence of inhibitors in prokaryotic genomes and suggested that this connection could serve as a more "straightforward path" to discovering more Acrs. Known CRISPR-Cas inhibitors have been identified either through the isolation of CRISPR-resistant phages or by proximity to anti-CRISPR associated genes. Some researchers have proposed looking for stable self-targeting CRISPR sequences as a potential indicator of genomes or mobile genetic elements harboring CRISPR inhibitors.
To test whether CRISPR inhibitors can be discovered systematically by flagging CRISPR self-targeting genomes, Doudna and her colleagues built a bioinformatic pipeline to search across the NCBI prokaryotic sequence database to locate self-targeting examples within predicted CRISPR arrays. The pipeline, which they called the Self-Targeting Spacer Searcher (STSS), first predicts all possible CRISPR arrays using the CRISPR Recognition Tool (CRT) and BLASTs each spacer against the host genome and any associated plasmids. Further, it collects information to gauge the likelihood that the self-targeting sequence would be lethal to the organism and if the target sequence occurs in a mobile genetic element.
They used their pipeline to collect self-targeting data for 150,291 genomes and observed 22,125 cases of predicted self-targets. Focusing initially on Pseudomonas aeruginosa, Listeria monocytogenes, and Neisseria meningitidis — in which multiple Acrs have been previously identified — the researchers determined the number of genomes that contained at least one lethal self-targeting CRISPR spacer, and the number of those genomes that also contained an Acr. In N. meningitidis, this was only 6 percent, while in P. aeruginosa and L. monocytogenes, it was more than 80 percent or 90 percent.
The researchers then looked to determine whether they could find new Acrs by screening genes in genomes containing self-targeting spacers, focusing their efforts on Cas12a, which had up to that point eluded discovery of inhibitory proteins. From the STSS results, they identified four strains of Moraxella bovoculi that contained self-targeting Cas12a systems as top candidates for containing anti-CRISPRs.
Through a series of screening experiments, the researchers discovered three inhibitors they called AcrVA1, AcrVA4, and AcrVA5. They further observed that AcrVA1 inhibited DNA cleavage by MbCas12a, LbCas12a, and AsCas12a, with the strongest inhibition observed for MbCas12a and weakest observed for AsCas12a. AcrVA4 and AcrVA5 inhibited double-stranded DNA cleavage for both MbCas12a and LbCas12a, but did not inhibit AsCas12a. They also noted that none of the AcrVA proteins inhibited S. pyogenes Cas9 cleavage.
"Together, these results establish a new approach for systematic discovery and validation of CRISPR-Cas inhibitors hidden within self-targeting genomes. Importantly, the Cas12a inhibitors revealed by this approach are only found within a few genomes within the NCBI database, with AcrVA4 and AcrVA5 being particularly rare genes, only co-occurring with each other," the authors wrote.
In a companion paper, researchers from UCSF and Massachusetts General Hospital noted that while Acrs have to date been discovered for Type I-D, I-E, I-F, II-A, and II-C CRISPR systems, their systematic discovery strategy yielded 12 more Acrs for Type I-C and Type V-A systems as well. Notably, one such protein they dubbed AcrVA1 was found to inhibit a wide variety of Cas12a orthologs, including MbCas12a, Mb3Cas12a, AsCas12a, and LbCas12a.
The researchers engineered P. aeruginosa to target phage JBD30 with type I-C CRISPR-Cas and used it in parallel with existing type I-E and I-F CRISPR strains to screen for additional Acrs. They then used a Type I-F inhibitor called acrIF11, which is commonly represented in both the P. aeruginosa mobilome and in more than 50 species of Proteobacteria, to discover Acr proteins in CRISPR systems where they have not yet been found: type I-C, a minimal Class 1 system, and type V-A CRISPR-Cas12a.
To find AcrIC and AcrVA proteins, the team searched for genomes encoding CRISPR spacers that match a target protospacer elsewhere in the same genome, and then used a strategy similar to that employed by Doudna's team.
"Although these two CRISPR subtypes do not share any protein components, a dual-specificity inhibitor may use distinct protein interaction interfaces or modulate an undiscovered host process required for CRISPR immunity," the authors wrote. "In sum, we used the anti-CRISPR 'key' acrIF11 to unlock Acr loci encoding seven distinct Acr genes inhibiting type I-C, I-F, and V-A CRISPR."
They also noted that AcrVA1 could serve as a tool for Cas12a regulation given how potently it inhibits the enzyme in bacteria and in human cells. "Our findings show that mobile genetic elements can tolerate bacteria with more than one CRISPR-Cas type by possessing multiple Acr proteins in the same locus," the team added. "The strategy described herein enabled the identification of many widespread anti-CRISPR proteins, which may prove useful in future anti-CRISPR discovery."