In nature, the best-known CRISPR system, CRISPR-Cas9, cuts any RNA or DNA it recognizes as foreign, and thereby protects bacteria from viral attacks. Another CRISPR system, one that is relatively obscure, protects bacteria in an entirely different way. Whereas CRISPR-Cas9 acts like a pair of molecular scissors, this alternative CRISPR system, CRISPR-Cas10, acts more like a molecular fumigator.

When CRISPR-Cas10 is activated in an infected bacterium, it instigates a chain of molecular events that culminates in a toxic cloud of inosine triphosphate (ITP). The ITP, which is generated via the deamination of adenosine triphosphate (ATP), stifles the bacterium’s growth, preventing phages from propagating to the bacterium’s fellows. Basically, the bacterium is sacrificed, but the bacterial population is preserved.

The details of the fumigation mechanism were uncovered by researchers headed by Dinshaw Patel, PhD, and Luciano Marraffini, PhD. Patel is the Abby Rockefeller Mauze Chair of Experimental Therapeutics at the Memorial Sloan Kettering Cancer Center, and Marraffini is head of the Laboratory of Bacteriology at Rockefeller University.

Patel, Marraffini, and colleagues presented their findings in Cell, in an article titled, “The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity.”

“Cas10 [triggers] two activities: single-stranded DNA degradation and synthesis of second messengers known as cyclic oligoadenylates (cOAs). The first activity is catalyzed by the HD domain of Cas10 and is sufficient to provide anti-phage immunity when the target viral transcript is expressed early, but not late, in the lytic cycle,” the article’s authors wrote. “By contrast, cOA production is carried out by the Palm domain of Cas10 to create a 3′–5′ cOA molecule using ATP as substrate (with cA4 and cA6 being the most abundant species), and it is essential for defense when the crRNA is complementary to a late-expressed phage RNA.”

In the current study, the researchers focused on the second activity, that is, the synthesis of cOAs. Some aspects of this activity were already fairly well understood. For example, Cas10 has been known to work through pathways similar to mammalian innate immunity pathways that produce cyclic nucleotides to activate a host response. But part of the Cas10 pathway involved a newly recognized CRISPR protein, CRISPR-associated adenosine deaminase 1 (Cad1). How, exactly, was Cad1 involved in stifling a bacterium’s activity? Part of the answer, the researchers determined, is that immunity is carried out through the activation of CRISPR-associated Rossman fold (CARF) effectors by the cOA second messenger.

“[We] characterized the function and structure of an effector in which the CARF domain is fused to [Cad1],” the article’s authors reported. “We show that upon binding of [the cOAs] cA4 or cA6 to its CARF domain, Cad1 converts ATP to ITP, both in vivo and in vitro. Cryoelectron microscopy structural studies on full-length Cad1 reveal an hexameric assembly composed of a trimer of dimers, with bound ATP at inter-domain sites required for activity and ATP/ITP within deaminase active sites. Upon synthesis of cAn during phage infection, Cad1 activation leads to a growth arrest of the host that prevents viral propagation.”

Graphical abstract from “The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity.” [Baca CF, Majumder P, Hickling JH, et al. Cell. DOI: 10.1016/j.cell.2024.10.002.]
According to co-first author and Marraffini laboratory member Christian F. Baca, the timing of the two-part response could be described as follows: “Cas10 alone can clear a phage or plasmid from a cell as long as the target transcript that’s been recognized by the guide RNA is made early in the viral infection. But if the problematic snippet is something only made at a later stage of the infection, these cOA molecules are essential for defense.”

A 3D structure of the full-length Cad1 protein, which researchers studied to explain how one CRISPR system responds to viruses. [Rockefeller University]
“The infected cell is sacrificed when the virus is sequestered within it, but the larger bacterial population is protected,” added co-first author and Patel laboratory member Puja Majumder. Why ITP is so toxic to the bacterium is unclear. One theory is that excess ITP competes for binding sites typically occupied by ATP or GTP in proteins that are critical for normal cellular function; another is that high levels of ITP interfere with phage DNA replication. “But we don’t really know why yet,” Majumder admitted.

One potential application of the scientists’ work is as a diagnostic tool for infection. “The presence of ITP,” Baca noted, “would indicate that a pathogen transcript is present in a sample.” In any event, the scientists’ finding indicates that CRISPR-Cas systems employ a wide range of molecular mechanisms beyond nucleic acid degradation to provide adaptive immunity in prokaryotes.

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