by Sarah Camara-Wilpert and David Mayo-Muñoz
Bacteria, just like humans, are frequently infected by viruses (bacteriophages or phages). Phages replicate at the expense of their hosts, killing them and the process releases hundreds of progeny into the environment to complete their life cycle. The end of bacteria? Not so fast! Bacteria evolved a variety of anti-phage defence mechanisms, such as the widespread CRISPR-Cas immunity. These systems store memories from previously infecting phages in the form of DNA snippets, which are stacked in between so-called CRISPR repeats. This CRISPR-memory array is turned into small RNA guides (CRISPR RNAs or crRNAs) that assemble with Cas proteins to recognize and destroy complementary phage nucleic acids during future infections.
You might be thinking, "Well, that's game over for the phages!" But hold on a second, turns out phages are no pushovers. In response to CRISPR-Cas, phages evolved anti-CRISPR (Acr) proteins that block CRISPR-Cas immunity at different stages. But is that really all? No! At the 10th anniversary of Acr discovery, we now add a new player to the game: RNA anti-CRISPRs or Racrs!
RNA anti-CRISPRs: A long-awaited revelation
In the realm of bacterial genomics, Kira Makarova and Eugene Koonin often foresee molecular phenomena years ahead of others in their field and this discovery was no exception. In 2019, using a bioinformatic approach, Koonin and colleagues discovered mysterious sequences in viral genomes that resembled single CRISPR repeats that were not associated with cas genes or arrays. They termed these solitary repeat units (SRUs) and proposed that phages repurposed them for alternative functions to CRISPR-Cas immunity. Intrigued by the possible role of these repeat mimics in CRISPR-Cas inhibition, we investigated a phage-encoded SRU resembling a type I-F CRISPR repeat. And indeed, this small RNA robustly blocked CRISPR-Cas immunity, hence we called it RacrIF1!
But how does it work? We found that phages use Racrs as decoys for the immune system components, thus allowing phages to continue replicating. RacrIF1 leads to the assembly of non-functional Cas complexes that do not carry all the crucial components for targeting the phage genome, titrating away Cas proteins from forming a functional targeting complex with host crRNAs.
We think this is a fascinating case. But is RacrIF1 the odd one out or are there any other RNAs that stop CRISPR-Cas? Excitingly, we not only identified Racrs in a wide range of phages and other mobile genetic elements infecting diverse prokaryotic taxa but also inhibiting diverse CRISPR-Cas systems. Even though it seems there's a large unexplored diversity of Racrs out there, it's important to stress that crRNA-like elements not only function as anti-CRISPRs (e.g. Varble et al., 2021; Workman et al., 2021; Li et al., 2021; and Shmakov et al., 2023), so it's crucial to explore these repeat mimics individually.
Molecular mimicry of immune components, a weak spot for immunity
In the intricate world of host-pathogen interactions, the phenomenon of molecular mimicry is a clever strategy employed by viruses to subvert host defenses beyond CRISPR during infection. Think of it as a high-stakes game of disguises at the molecular level, where viruses really perfected the art of disguise. For instance, some phages mimic antitoxin non-coding RNAs or host methyltransferases to ensure viral replication in the face of anti-phage defenses. Also, human viruses can hijack our defense components, such as tumor suppressors or inhibitors of the complement system. It's akin to these viruses co-opting immune components to further their own agenda.
Shaking up the biotech scene? Maybe one day!
Racr similarity to CRISPR repeats simplifies identification (vs. Acr proteins) and offers potential for rational design, which makes them promising candidates for future biotech applications. For example, Racrs could serve as off-switches for CRISPR-Cas-based tools potentially revolutionizing genome editing technologies. Additionally, phage therapy, offering a promising alternative to antibiotics in the face of rising antibiotic resistance, could be improved by decorating phage genomes with inhibitors such as Racrs to overcome the defenses of the targeted pathogen.
Collaborating overseas for 4 years
Ours were not only extraordinary findings, the process of obtaining them was great fun. This work was the result of a fabulous collaboration between outstanding teams spanning hemispheres, led by Rafael Pinilla-Redondo, Assistant Professor at the University of Copenhagen (Denmark), and Peter Fineran, Professor at the University of Otago (New Zealand). We exchanged knowledge, strains, wild ideas, and endless encouragement for almost four years!
We (Sarah and David) made a great team as co-first authors during our PhD studies, with many Zoom meetings at odd hours of the day. After many years of working far apart but closely together, we finally had a reunion in Tbilisi, Georgia at VOM2023 this year! Currently, David is an assistant research fellow at the Phi Lab at the University of Otago (New Zealand), and Sarah is about to transition into industry as a Development Scientist for Chr. Hansen (Denmark).