It serves as a kind of molecular fumigator to combat phages and plasmids.
CRISPR-Cas9 has been frequently compared to a pair of genetic scissors because of its ability to precisely cut out specific DNA segments.
However, research reveals that CRISPR systems utilize various strategies in their defense toolkit. Initially found in bacteria, where it has served as an adaptive immune system for millions of years, CRISPR is naturally used by some single-celled organisms to defend against viruses (referred to as phages) and other foreign genetic materials. Recently, researchers from Rockefeller’s Laboratory of Bacteriology, led by Luciano Marraffini, and the Structural Biology Laboratory at MSKCC, led by Dinshaw Patel, uncovered how a specific CRISPR system not only employs genetic scissors but also functions as a form of molecular fumigator. In their latest study published in Cell, the scientists revealed that this system, known as CRISPR-Cas10, inundates a virally infected bacterium with toxic molecules to prevent the virus from spreading to surrounding bacterial cells.
“This introduces a completely new form of CRISPR chemistry,” says co-first author Christian Baca, a graduate student from the TPCB in Marraffini’s lab. “It further demonstrates that CRISPR systems possess a variety of immune responses.”
Cell Shutdown
There are six varieties of CRISPR (“clustered regularly interspaced short palindromic repeats”) systems; for instance, CRISPR-Cas9 represents type II, where the enzyme Cas9 operates as the DNA cutter. In the current research, the focus was on a type III system called CRISPR-Cas10.
In both the CRISPR-Cas9 and CRISPR-Cas10 systems, guide RNAs pinpoint troublesome genetic material, prompting the enzymes to start cutting. However, the CRISPR-Cas10 complex additionally generates a surge of small second messenger molecules known as cyclic-oligoadenylates (cOAs), which contribute to halting cellular processes, thereby curbing viral spread. This dual approach can be likened to fumigating an infested room and quickly closing the door to prevent the problem from extending to other areas.
This two-pronged response heavily relies on timing, according to Baca. “Cas10 can eliminate a phage or plasmid from a cell as long as the target transcript recognized by the guide RNA is produced early in the viral infection. However, if the troublesome snippet appears later in the infection process, the cOA molecules become critical for defense,” he explains. “In this manner, type III CRISPR systems operate similarly to mammalian innate immunity pathways like cGAS-STING, which generate cyclic nucleotides to trigger a host response,” Marraffini adds.
While these mechanisms were known, the intricate molecular processes by which a new Type III CRISPR protein, known as CRISPR-associated adenosine deaminase 1 (Cad1), induces cell shutdown remained unclear.
A Toxic Plume
To investigate this, the researchers conducted a comprehensive molecular and structural examination of Cad1, utilizing cryo-electron microscopy (cryo-EM) and other sophisticated techniques to uncover unique structures and behaviors that elucidate how the system pauses cellular function.
In the CRISPR-Cas10 system, Cad1 is activated by the binding of cOAs to a region of the protein designated as the CARF domain. This interaction prompts Cad1 to convert ATP (the cell’s energy currency) into ITP (an intermediary nucleotide that is usually present in minimal amounts in the cell), which subsequently saturates the cell. When ITP accumulates to high levels, it becomes toxic to cells, causing a cessation of cellular activity and inducing a dormant state.
“The infected cell is sacrificed while the virus is contained within, but the larger bacterial community is safeguarded,” states co-first author Puja Majumder, a postdoctoral research scholar at the Patel Lab. The reason behind this effect remains uncertain. One hypothesis suggests that excessive ITP competes for binding sites that are typically filled by ATP or GTP in proteins essential for normal cellular function, while another proposes that high ITP levels disrupt phage DNA replication.
“But we don’t have a definitive answer yet,” Majumder admits.
A potential application of their findings lies in developing a diagnostic tool for infections, as Baca points out. “The presence of ITP could indicate that a pathogen transcript is found within a sample.”
