CRISPR-Cas9 has long been likened to a pair of genetic scissors because of its ability to elegantly and precisely snip any desired DNA fragment. But it turns out that the CRISPR system has more than just one strategy in its toolbox. CRISPR is a mechanism originally discovered in bacteria, and it has been operating for centuries as an adaptive immune system. Certain single-celled organisms naturally use CRISPR to protect themselves from viruses (called bacteriophages) and other foreign genetic fragments.
Now, in a new study, Luciano Marraffini and his team at the Rockefeller University Bacteriology Laboratory and Dinshaw Patel and his team at the Memorial Sloan-Kettering Cancer Center Structural Biology Laboratory have discovered a CRISPR system that not only uses genetic scissors to deal with invaders, but also acts as a kind of molecular fumigant. The relevant research results were recently published in the journal Cell, with the title of the paper "The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity". In the paper, the authors show that this CRISPR system, called CRISPR-Cas10, can flood virus-infected bacteria with toxic molecules, thereby preventing the virus from spreading to the rest of the bacterial population. This is a completely new type of CRISPR chemistry. This further demonstrates that CRISPR systems have a range of immunity strategies.
There are six types of CRISPR systems. For example, CRISPR-Cas9 belongs to the Type II CRISPR system, where the Cas9 enzyme is a DNA scissor. In the new study, the authors studied a Type III CRISPR system called CRISPR-Cas10.
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In both systems, a guide RNA (gRNA) identifies the problematic genetic material, and then the enzyme begins cutting. However, the CRISPR-Cas10 complex also produces a small second messenger molecule called cyclic oligoadenylate (cOA) that helps shut down cellular activity and thus prevent the virus from spreading. This second line of defense is like fumigating a room infested with pests and then quickly closing the door to contain the infestation and prevent it from spreading to other parts of the house.
"This two-pronged approach depends very much on timing," says co-first author Christian Baca of the Marraffini lab. "As long as the target transcript recognized by the gRNA is produced early in the viral infection, Cas10 alone can clear the cell of the phage or plasmid. But if the problematic fragment is produced later in the infection, then these cOA molecules are essential for defense."
"In this way, the type III CRISPR system works similarly to mammalian innate immune pathways such as cGAS-STING, which produce cyclic nucleotides to activate host responses," Marraffini adds.
While this much is known, the exact molecular dynamics behind how a new type III CRISPR protein called CRISPR-associated adenosine deaminase 1 (Cad1) achieves cellular shutdown were not.
To find out, the authors performed a detailed molecular and structural analysis of Cad1, using cryo-electron microscopy and other advanced methods to reveal unusual structures and dynamics that explain how the system pauses cellular activity.
In the CRISPR-Cas10 system, Cad1 alerts the virus to its presence by binding to a part of the protein called the CARF domain via cOA. This in turn stimulates Cad1 to convert ATP into ITP, an intermediate nucleotide that is normally present in small amounts in cells, as ITP floods the cell. High doses of ITP are toxic to cells, so cellular activity stops and the cell becomes dormant.
Figure 1. Schematic diagram of the working process of CRISPR-Cas10 system. (Baca C F, et al., 2024)
"When the virus is sequestered inside the cell, the infected cell is sacrificed, but the larger bacterial population is protected," said Puja Majumder, a postdoctoral research scholar in Patel's lab and co-first author of the paper. It's not clear why it has this effect. One theory is that excess ITP competes for binding sites normally occupied by ATP or GTP in proteins that are critical for normal cellular function. Another theory is that high levels of ITP interfere with the replication of phage DNA.
One potential application of their discovery is as a diagnostic tool for infection. "The presence of ITP indicates the presence of pathogen transcripts in the sample," Baca noted.
Reference
Baca C F, et al. The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity. Cell, 2024.