Research Newsletter | Fall 2025

While completing her thesis, Nicole became increasingly fascinated with another ancient arms race—between bacteria and their viruses (phage). During her studies, CRISPR-Cas had been discovered as the first adaptive immune system in bacteria. CRISPR-Cas systems acquire short snippets of the phage genome, which they use to provide immunological memory against later infection. The discovery that bacteria, like humans, have both innate and adaptive immunity underscored how little was known about bacterial defense mechanisms. Studies demonstrating that Cas9 can cut specified DNA sequences in vitro and in human cells launched a “CRISPR craze” that revolutionized molecular biology and gene therapy. Shortly afterwards, Dr. Joseph Bondy-Denomy discovered the first phage-encoded “anti-CRISPR” proteins that inhibit CRISPR-Cas systems. Inspired by the enormous potential for new discoveries—and their broad impacts on medicine and biotechnology—Nicole decided to explore anti-CRISPRs in Joseph Bondy-Denomy’s lab at the University of California, San Francisco as a postdoctoral fellow. She set out to discover the first anti-CRISPRs that inhibit Cas12a, a newly discovered CRISPR-Cas nuclease that was also co-opted for gene editing. Using bioinformatics and bacterial models, Nicole discovered the first Cas12a anti-CRISPRs in a phage infecting Moraxella bovoculi, a bovine pathogen. One of these anti-CRISPRs, AcrVA1, inhibits Cas12a in vitro and in human cells, which enables it to regulate Cas12a during gene editing.

In more recent work, Nicole and collaborators found that another anti-CRISPR (AcrVA2) inhibits Cas12a using a novel mechanism for molecular conflict. Surprisingly, AcrVA2 interrupts Cas12a biogenesis by binding to its N-terminal polypeptide and triggering destruction of its mRNA before translation is complete. This polypeptide-dependent mRNA downregulation achieves two feats: first, it allows AcrVA2 to broadly recognize conserved amino acids that are present in otherwise divergent Cas12a orthologs. Second, this mechanism appears to constrain Cas12a escape. Amino acid mutations in Cas12a that prevent binding and downregulation by AcrVA2 also compromise Cas12a anti-phage activity, indicating that AcrVA2 evolved to recognize features that Cas12a cannot afford to lose. Structural and phylogenetic analyses suggest that polypeptide-dependent mRNA downregulation is widespread across bacteria and may impact other aspects of bacterial physiology, including the spread of antibiotic resistance.

Future research in the Marino laboratory will explore polypeptide-dependent mRNA downregulation and identify other targets in bacteria. They will use biochemical, genetic, and structural approaches to reveal how this anti-CRISPR recognizes the Cas12a polypeptide—perhaps as it emerges from the ribosome—and triggers destruction of its mRNA. By understanding this mechanism further, her lab hopes to unlock a novel therapeutic paradigm. The ability to recognize broadly conserved features while limiting resistance makes polypeptide-dependent mRNA downregulation a promising antiviral strategy. By recognizing specific amino acids, rather than a conserved stretch of nucleotide sequence, polypeptide-dependent mRNA downregulation may better enable broad-spectrum targeting of divergent viruses while limiting viral resistance. More broadly, her lab is focused on understanding how phages overcome multilayered bacterial defenses to improve phage therapy. Multidrug-resistant bacterial infections pose one of the greatest threats to human health worldwide. Phage have been used successfully to treat multidrug resistant infections or resensitize bacteria to antibiotics. Phages are attractive as antimicrobials because of their high specificity for a bacterial species or strain, which enables them to clear the infection without disrupting the microbiome. However, to successfully replicate and lyse their host, phage must first disarm or evade bacterial defenses. While over a hundred antiphage defense systems have been discovered in bacteria over the past several years, it remains unclear how phages overcome or evade most of these defenses. Understanding how phages achieve this feat will be critical for unlocking their potential for human health.