A new chemistry for CRISPR

But it turns out that CRISPR systems have more than one strategy in their toolkit. A mechanism originally discovered in bacteria, where it has operated as an adaptive immune system for eons, CRISPR is naturally deployed by certain singled-cell organisms to protect themselves against viruses (called phages) and other foreign genetic fragments. Now, researchers at Rockefeller’s Laboratory of Bacteriology headed by Luciano Marraffini, and at the MSKCC’s Structural Biology Laboratory headed by Dinshaw Patel, have discovered how one CRISPR system battles invaders with not only genetic scissors but also acts as a sort of molecular fumigator. In a recent publication in Cell, the scientists found that this system, called CRISPR-Cas10, floods a virally infected bacterium with toxic molecules, and thus prevents the virus from spreading through the rest of the bacterial population.

“It’s a completely brand-new type of CRISPR chemistry,” says co-first author Christian Baca, a TPCB graduate student in the Marraffini lab. “It’s more evidence that CRISPR systems have an array of immune strategies at their disposal.”

Cell shutdown

There are six types of CRISPR (“clustered regularly interspaced short palindromic repeats”) systems; CRISPR-Cas9, for example, is type II, with the enzyme Cas9 functioning as the DNA scissors. For the current study, the researchers looked at a type III system called CRISPR-Cas10.

In both systems, guide RNAs identify problematic genetic material, and the enzymes begin snipping. However, the CRISPR-Cas10 complex also produces a burst of small second messenger molecules called cyclic-oligoadenylates (cOAs), which helps shut down cell activity, thereby preventing the virus from spreading. This second line of attack is akin to fumigating one pest-ridden room, and then quickly shutting the door to keep the infestation contained so it can’t spread to the rest of the house.

This two-part response is largely a matter of timing, says Baca. “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,” he says. “In this way, type III CRISPR systems work similarly to mammalian innate immunity pathways, such as cGAS-STING, that produce cyclic nucleotides to activate a host response,” adds Marraffini.

While that much was known, the molecular dynamics behind how, exactly, a new Type III CRISPR protein, CRISPR-associated adenosine deaminase 1 (Cad1) achieves cell shut down was not.

A toxic plume

To find out, the researchers undertook a detailed molecular and structural analysis of Cad1, using cryo-EM and other advanced approaches to reveal unusual structures and dynamics that explain how the system pauses cell activity.

In the CRISPR-Cas10 system, Cad1 is alerted to the presence of a virus by the binding of cOAs to a part of the protein called the CARF domain. That in turn stimulates Cad1 to convert ATP (the energy currency of the cell) into ITP (an intermediary nucleotide that usually is present in the cell in small amounts), which then floods the cell. ITP turns toxic to cells in high doses, so as a result, cellular activity comes to a halt, putting the cell in a dormant state.

“The infected cell is sacrificed when the virus is sequestered within it, but the larger bacterial population is protected,” says co-first author Puja Majumder, a postdoctoral research scholar at the Patel Lab. Why it has this impact 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 says.

One potential application of their discovery is as a diagnostic tool for infection, Baca notes. “The presence of ITP would indicate that a pathogen transcript is present in a sample.”