Unveiling the Molecular Mechanisms of CRISPR-Cas10's Cellular Shutdown

CRISPR-Cas9 can elegantly and precisely cut away any desired region of DNA and has long been compared to a type of genetic scissors.

However, it turns out that CRISPR systems have a variety of strategies at their disposal. Certain single-cell organisms naturally use CRISPR, a technique first identified in bacteria where it has functioned for generations as an adaptive immune system, to defend themselves against viruses (also known as phages) and other foreign genetic pieces.

Researchers at the MSKCC's Structural Biology Laboratory, led by Dinshaw Patel, and Rockefeller's Laboratory of Bacteriology, led by Luciano Marraffini, have now figured out how one CRISPR system fights intruders by acting as a kind of molecular fumigator in addition to genetic scissors.

This mechanism, known as CRISPR-Cas10, bombards a virus-infected cell with harmful chemicals, stopping the virus from spreading to the rest of the bacterial population, according to a recent study published in the journal Cell.

It is a completely brand-new type of CRISPR chemistry. It is more evidence that CRISPR systems have an array of immune strategies at their disposal.”

Christian Baca, Study Co-First Author and Graduate Student, Rockefeller University

Cell Shutdown

CRISPR (or “clustered regularly interspaced short palindromic repeats”) systems come in six varieties. CRISPR-Cas9, for instance, is type II, using the enzyme Cas9 as the DNA scissors. For the current investigation, the researchers examined a type III system known as CRISPR-Cas10.

In both systems, the enzymes start snipping when guide RNAs recognize faulty genomic material. But the CRISPR-Cas10 complex also generates cyclic-oligoadenylates (cOAs), which are tiny second messenger molecules that assist in stopping cell activity and prevent the virus from spreading.

This second approach is like fumigating a pest-infested room and then swiftly sealing the door to contain the infestation, preventing it from spreading throughout the house.

According to Baca, this two-part response mostly depends on timing.

Cas10 alone can clear a phage or plasmid from a cell as long as the target transcript that is recognized by the guide RNA is made early in the viral infection. However, if the problematic snippet is something only made at a later stage of the disease, these cOA molecules are essential for defense. 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.”

Luciano Marraffini, Rockefeller University

Although this much was known, nothing was understood about the molecular dynamics underlying the precise mechanism by which CRISPR-associated adenosine deaminase 1 (Cad1), a novel Type III CRISPR protein, causes cell shutdown.

A Toxic Plume

Using cryo-EM and other cutting-edge techniques, the researchers conducted a thorough molecular and structural investigation of Cad1 to uncover odd structures and dynamics that explain how the system halts cell activity.

The binding of cOAs to a region of the protein known as the CARF domain in the CRISPR-Cas10 system alerts Cad1 to the presence of a virus. The cell is subsequently flooded with ITP, an intermediary nucleotide that is typically present in trace levels, after Cad1 has been stimulated to convert ATP, the cell's energy currency, into ITP. High concentrations of ITP cause cells to become toxic, which stops cellular activity and puts the cell in a dormant condition.

“The infected cell is sacrificed when the virus is sequestered within it, but the larger bacterial population is protected,” said Puja Majumder, Co-First Author and Postdoctoral Research Scholar at the Patel Lab.

It is unclear why it has this effect. One explanation is that too much ITP interferes with phage DNA replication, while another is that it competes for binding sites in proteins that ATP or GTP normally occupies.

“But we do not really know why yet,” Majumder said.

According to Baca, one possible use for their discovery is as an infection diagnostic tool. “The presence of ITP would indicate that a pathogen transcript is present in a sample.”