AcaFinder: Genome Mining for Anti-CRISPR-Associated Genes

Area of Science:

• Genomics and Bioinformatics
• Microbial Genetics
• Molecular Biology

Background:

• Anti-CRISPR (Acr) proteins inhibit host CRISPR-Cas systems, often co-occurring with Acr-associated (Aca) proteins in acr-aca operons.
• Existing bioinformatics tools primarily focus on Acr discovery, leaving Aca identification unaddressed.
• Aca proteins act as self-transcriptional repressors of acr-aca operons, functioning as anti-anti-CRISPRs with implications for CRISPR-Cas technology.

Purpose of the Study:

• To develop the first bioinformatics tool, AcaFinder, for automated genome mining of Aca proteins and their associated acr-aca operons.
• To identify novel Aca families and analyze the evolutionary relationships between Acr and Aca proteins within operons.

Main Methods:

• AcaFinder utilizes guilt-by-association (GBA) to predict Acas and acr-aca operons, alongside Hidden Markov Model (HMM) database searches for known Aca homologs.
• The tool analyzes input genomes for prophages, CRISPR-Cas systems, and self-targeting spacers (STSs).
• AcaFinder was applied to over 16,000 prokaryotic and 142,000 gut phage genomes, followed by multistep filtering.

Main Results:

• AcaFinder identified 36 high-confidence new Aca families, a threefold increase over the 12 previously known families.
• Seven new Aca families were discovered in major human gut bacteria (Bacteroidota, Actinobacteria, Fusobacteria) and their phages, expanding the known host range.
• Analysis revealed a complex association network between Acrs and Acas, indicating frequent recombination events forming diverse acr-aca operon combinations.

Conclusions:

• AcaFinder is an effective tool for discovering novel Aca proteins and understanding their genomic context.
• The study significantly expands the repertoire of known Aca families and highlights their prevalence in diverse bacterial and phage communities.
• The findings underscore the evolutionary plasticity of acr-aca operons and the potential of Aca proteins in modulating CRISPR-Cas immunity and biotechnological applications.