Feussner, M. | Search bioRxiv

bioRxiv2026

An expanded realm of anti-CRISPR-associated proteins and regulatory mechanisms

Many bacteriophages encode anti-CRISPR (Acr) proteins that inhibit the CRISPR-Cas immune systems. Rapid acr gene expression upon phage entry enables CRISPR-Cas neutralisation, but can impact phage fitness if unregulated. Therefore, Acr production is often controlled by distinct families of co-encoded anti-CRISPR-associated (Aca) proteins, which are usually helix-turn-helix (HTH) regulators that bind DNA within acr-aca operon promoters. Previously, we demonstrated that the Aca2 family additionally represses Acr production translationally by binding structured RNA motifs within the 5' untranslated regions (UTRs) of the acr-aca mRNA. Here, through systematic bioinformatic analyses, we provide evidence of structured RNA motifs in the 5' UTRs of operons encoding members of other Aca families, and that Aca1 also specifically binds its cognate RNA motif. Additionally, many Aca proteins are predicted to regulate not only their own but also adjacent operons with potential anti-defence genes. Indeed, we show that Aca14, newly identified in this study, represses two predicted anti-defence operons. Aca14 is a ribbon-helix-helix domain protein, revealing regulatory diversity beyond the canonical HTH Aca family members. Collectively, our findings expand the understanding of acr regulation in mobile genetic elements and reveal novel mechanisms by which phages fine-tune anti-defence gene expression. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/740549v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@23e8edorg.highwire.dtl.DTLVardef@19146a8org.highwire.dtl.DTLVardef@b5e553org.highwire.dtl.DTLVardef@1e2ddbf_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2026

Phage-encoded single-guide RNAs subvert CRISPR-Cas9 immunity

Central to CRISPR technologies is the single-guide RNA (sgRNA), an engineered fusion of a processed CRISPR RNA and tracrRNA that directs Cas9 and many Cas12 nucleases to bind and cleave target DNA1-4. Here, we report the discovery of similarly compact viral sgRNAs (vsgRNAs) encoded by bacteriophages that counteract bacterial CRISPR-Cas9 immunity. vsgRNAs inhibit Cas9 function via two complementary routes: by sequestering the Cas9 apoenzyme, and by re-directing the Cas9 nuclease to transcriptionally silence its own promoter. vsgRNAs also can cooperate with co-encoded anti-CRISPR proteins (Acrs), including AcrIIA25.1 that blocks DNA binding by Cas9 complexed with a standard sgRNA but not with the vsgRNA. We predict that phages evolved vsgRNAs by co-opting and repurposing host-encoded long-form tracrRNAs (tracr-L) responsible for Cas9 auto-repression and a countermeasure to Acrs5,6. Our search also uncovered Cas9-regulating small CRISPR-associated RNAs (scaRNAs), which we predict were inserted upstream of tracrRNAs to form tracr-L but were also co-opted by phages as viral scaRNAs to suppress Cas9 immunity. Finally, we found that vsgRNAs can enable genome editing in mammalian cells, offering a natural guide RNA template for CRISPR technologies. Overall, these findings reveal that bacteriophages devised their own compact sgRNAs tailored to subvert Cas9 immunity, long preceding their rational design to program RNA-guided nucleases2.

microbiology↗