It’s Capable of Even More Than We Thought: Scientists Discover CRISPR’s New Secret Weapon

A newly discovered bacterial defense protein named Cat1 reveals an extraordinary method of viral neutralization.

All living things, from animals to the tiniest microbes, need ways to protect themselves. Bacteria may be small and simple, but they’ve evolved incredibly clever strategies to fight off viral attackers. One of their best-known defense tools is CRISPR-Cas9, which scientists have repurposed into a groundbreaking gene-editing technology approved by the FDA.

Over the past year, researchers at Rockefeller University and Memorial Sloan Kettering Cancer Center have been exploring another layer of this microbial defense system. Led by Luciano Marraffini and Dinshaw Patel, the teams focused on a special group of immune proteins known as CARF effectors. These molecular tools help bacteria shut down infected cells, stopping viruses from spreading to nearby cells.

In a recent study published in Science, the scientists revealed a powerful new CARF effector they named Cat1. What makes Cat1 so effective is its unusually complex structure. It targets and destroys a key molecule that cells need to function. Without that vital fuel, the invading virus is left stranded and unable to continue its attack.

“The collective work of our labs is revealing just how effective—and different—these CARF effectors are,” says Marraffini. “The range of their molecular activities is quite amazing.”

Multiple defense systems

CRISPR is a mechanism in the adaptive immune systems of bacteria and other certain single-cell organisms that offers protection against viruses, called phages. The six types of CRISPR systems work roughly the same way: A CRISPR RNA identifies foreign genetic code, which triggers a cas enzyme to mediate an immune response, often snipping off the invader material.

But an increasing body of evidence indicates that CRISPR systems deploy a wide variety of defensive strategies beyond genetic scissors. Marraffini’s lab has led the way on much of this research. In particular, they have been studying a class of molecules in CRISPR-Cas10 systems called CARF effectors, which are proteins that are activated upon phage infection of a bacterium.

Cat1 monomers seen here in shades of pink and purple, while cA4 gluing the dimers to extend the filament are in orange. Credit: Laboratory of Bacteriology at The Rockefeller University

CARF effector immunity is believed to work by creating an inhospitable environment for viral replication. For example, the Cam1 CARF effector causes membrane depolarization of an infected cell, while Cad1 triggers a sort of molecular fumigation, flooding an infected cell with toxic molecules.

Metabolic freeze

For the current study, the researchers wanted to try to identify additional CARF effectors. They used Foldseek, a powerful structural homology search tool, to find Cat1.

They found that Cat1 is alerted to the presence of a virus by the binding of secondary messenger molecules called cyclic tetra-adenylate, or cA₄, which stimulate the enzyme to cleave an essential metabolite in the cell called NAD⁺.

“Once a sufficient amount of NAD⁺ is cleaved, the cell enters a growth-arrest state,” says co-first author Christian Baca, a TPCB graduate student in the Marraffini lab. “With cellular function on pause, the phage can no longer propagate and spread to the rest of the bacterial population. In this way, Cat1 is similar to Cam1 and Cad1 in that they all provide population-level bacterial immunity.”

Unique complexity

But while its immune strategy may be similar to these other CARF effectors, its form is not, as co-first author Puja Majumder, a postdoctoral research scholar in the Patel Lab, revealed through detailed structural analysis using cryo-EM.

She found that the Cat1 protein has a surprisingly complex structure in which Cat1 dimers are glued by cA₄ signal molecule, forming long filaments upon viral infection, and trap the NAD⁺ metabolites within sticky molecular pockets. “Once the NAD⁺ metabolite is cleaved by Cat1 filaments, it’s not available for the cell to use,” Majumder explains.

But the protein’s singular structural complexity doesn’t stop there, she adds. “The filaments interact with each other to form trigonal spiral bundles, and these bundles can then expand to form pentagonal spiral bundles,” she says. The purpose of these structural components remains to be investigated.

Also unusual is the fact Cat1 often seems to work alone. “Normally in type III CRISPR systems, you have two activities that contribute to the immunity effect,” Baca says. “However, most of the bacteria that encode Cat1 seem to primarily rely on Cat1 for their immunity effect.”

Marraffini says these findings pose intriguing new questions. “While I think we’ve proven the big picture—that CARF effectors are great at preventing phage replication—we still have a lot to learn about the details of how they do it. It will be fascinating to see where this work leads us next.”

Reference: “Cat1 forms filament networks to degrade NAD+ during the type III CRISPR-Cas antiviral response” by Christian F. Baca, Puja Majumder, James H. Hickling, Dinshaw J. Patel and Luciano A. Marraffini, 10 April 2025, Science.
DOI: 10.1126/science.adv9045

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