Researchers discover CRISPR-Cas10 can flood virally infected bacteria with toxic molecules

CRISPR-Cas9 has long been likened to a kind of genetic scissors, thanks to its ability to cut any desired section of DNA with exquisite precision.

But it turns out that CRISPR systems have more than one strategy in their toolkit. CRISPR, a mechanism originally discovered in bacteria and working as an adaptive immune system for ages, is naturally deployed by certain single-celled organisms to protect themselves against viruses (called phages) and other foreign genetic fragments.

Researchers at the Rockefeller Laboratory of Bacteriology, headed by Luciano Marraffini, and the MSKCC Structural Biology Laboratory, headed by Dinshaw Patel, have discovered how a CRISPR system fights invaders not just with genetic scissors, but also as a type of molecular pest control tool.

One last release inside CellScientists found that this system, called CRISPR-Cas10, floods a virally infected bacterium with toxic molecules, thus preventing the virus from spreading to the rest of the bacterial population.

“This is a completely new type of CRISPR chemistry,” says co-author Christian Baca, a TPCB graduate student in the Marraffini lab. “This is further evidence that CRISPR systems have a range of immune strategies at their disposal.”

cell shutdown

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

In both systems, guide RNAs detect the problematic genetic material and enzymes begin to break it down. However, the CRISPR-Cas10 complex also produces a burst of small second messenger molecules called cyclic-oligoadenylates (cOAs) that help shut down cell activity, thus preventing the virus from spreading. This second line of attack is akin to disinfecting a room full of pests and then quickly closing the door to keep the infestation under control so it doesn’t spread to the rest of the house.

Baca says this two-part response is largely a matter of timing.

“Cas10 alone can clear a phage or plasmid from a cell as long as the target transcript recognized by the guide RNA is made at an early stage of viral infection. But if the offending particle is something that is only made at a later stage of infection, these cOA molecules are required for defense,” he says.

“In this way, type III CRISPR systems work similarly to mammalian innate immune pathways such as cGAS-STING, which produce cyclic nucleotides to activate a host response,” adds Marraffini.

This much was known, but what is certain molecular dynamics It is unknown how CRISPR-associated adenosine deaminase 1 (Cad1), a novel Type III CRISPR protein, manages to shut down the cell.

A poisonous feather

To find out, the researchers performed 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 cOAs binding to a portion of the protein called the CARF domain. This stimulates Cad1 to convert ATP (the cell’s energy currency) into ITP (an intermediate nucleotide usually found in small amounts in the cell), which then fills the cell. ITP becomes toxic cells When used in high doses, cellular activity stops and the cell enters a dormant state.

“The infected cell is sacrificed when trapped within the virus, but the larger bacterial population is preserved,” says co-first author Puja Majumder, a postdoctoral research scientist in the Patel Laboratory. It is unclear why it has this effect. One theory is that excess ITP competes for binding sites typically occupied by ATP or GTP in proteins critical for normal cellular function; Another is that high ITP levels interfere with phage DNA replication.

“But we don’t know exactly why yet,” says Majumder.

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