Target RNA-guided protease activity in type III-E CRISPR–Cas system

Abstract

The type III-E CRISPR–Cas systems are newly identified adaptive immune systems in prokaryotes that use a single Cas7–11 protein to specifically cleave target RNA. Cas7–11 could associate with Csx29, a putative caspase-like protein encoded by the gene frequently found in the type III-E loci, suggesting a functional linkage between the RNase and protease activities in type III-E systems. Here, we demonstrated that target RNA recognition would stimulate the proteolytic activity of Csx29, and protein Csx30 is the endogenous substrate. More interestingly, while the cognate target RNA recognition would activate Csx29, non-cognate target RNA with the complementary 3′ anti-tag sequence inhibits the enzymatic activity. Csx30 could bind to the sigma factor RpoE, which may initiate the stress response after proteolytic cleavage. Combined with biochemical and structural studies, we have elucidated the mechanisms underlying the target RNA-guided proteolytic activity of Csx29. Our work will guide further developments leveraging this simple RNA targeting system for RNA and protein-related applications.

Nucleic Acid Detection through RNA‐Guided Protease Activity in Type III‐E CRISPR‐Cas Systems

“…Time‐dependent fluorescence intensity increases in the supernatant fractions, correlating with Csx30 cleavage, were observed under all the tested conditions (Figure 1E–G). Particularly, the Craspase system with catalytic dead Cas7‐11 (D547A/D698A) [7b] was found to be more efficient in catalyzing Csx30 cleavage and fluorescence signal increase (Figure 1F), in line with the previous report [12] . Craspase containing catalytic dead Cas7‐11 was therefore used for the nucleic acid detection assays unless otherwise stated.…”

“…The crRNAs complementary to specific regions of the genes were generated for the reconstitution of Craspase‐based detection systems (Figure 2A). The type III‐E system uses a 2‐nt 3’‐tag in the upstream of the spacer to distinguish self (with complementary 3’‐anti‐tag) and non‐self ssRNA (with non‐complementary 3’‐anti‐tag) [12] . Csx29 bound to the tail of the Cas7‐11‐crRNA complex was activated through a conformational change triggered by the recognition of non‐self target RNA but not that with complementary 3’‐anti‐tag [7] .…”

“…Notably, the Craspase systems show species‐specific substrate cleavage specificity [12] . For instance, upon activation by target RNA, Craspase (Csx29) from S. brodae (Sb) could cleave SbCsx30 substrate but not Csx30 from D. ishimotonii (DiCsx30) and vice versa.…”

Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox

“…10,33 These components form the basis of a system for a RNA-triggered protease system, where Cas7-11 effector binding activates the associated Csx29 protease to cleave the Csx30 substrate. 34−37 This cleavage generates an N-terminal product, which affects host growth though a RpoE stressassociated response 35,36,38 Class 2 consists of CRISPR types II, V, and VI (Figure 2). As mentioned above, the defining feature of class 2 is that their effector modules comprise a single, large, multidomain protein, which makes them the obvious tools of choice for genome editing and other applications.…”

“…The evolution of III-E CRISPR systems from III-D also involved the loss of other cas genes, in particular the effector DNA endonuclease encoded by cas10 , resulting in the loss of DNA cleavage and a transition to exclusively RNA targeting . In addition, during their evolution, the type III-E systems acquired the CHAT protease Csx29 and its corresponding substrate Csx30, as well as the accessory protein Csx31 and σ factor RpoE. , These components form the basis of a system for a RNA-triggered protease system, where Cas7-11 effector binding activates the associated Csx29 protease to cleave the Csx30 substrate. − This cleavage generates an N-terminal product, which affects host growth though a RpoE stress-associated response ,, …”