Structural insights into the modulatory role of the accessory protein WYL1 in the Type VI-D CRISPR-Cas system

Abstract

The Type VI-D CRISPR-Cas system employs an RNA-guided RNase Cas13d with minimal targeting constraints to combat viral infections. This CRISPR system contains RspWYL1 as a unique accessory protein that plays a key role in boosting its effector function on target RNAs, but the mechanism behind this RspWYL1-mediated stimulation remains completely unexplored. Through structural and biophysical approaches, we reveal that the full-length RspWYL1 possesses a novel three-domain architecture and preferentially binds ssRNA with high affinity. Specifically, the N-terminus of RspWYL1 harbors a ribbon-helix-helix motif reminiscent of transcriptional regulators; the central WYL domain of RspWYL1 displays a Sm-like β-barrel fold; and the C-terminal domain of RspWYL1 primarily contributes to the dimerization of RspWYL1 and may regulate the RspWYL1 function via a large conformational change. Collectively, this study provides a first glimpse into the complex mechanism behind the RspWYL1-dictated boosting of target ssRNA cleavage in the Type VI-D CRISPR-Cas system.

A Sensitive, Rapid, and Portable CasRx-based Diagnostic Assay for SARS-CoV-2

“…Like LwaCas13a, Cas13d is more flexible than most other Cas enzymes because it lacks a protospacer flanking sequence (PFS) requirement 28,44,46 , permitting targeting of any sequence without constraint. In addition, some native Cas13d systems include a WYL1-domain-containing accessory protein, which has been demonstrated to increase the on-target and collateral cleavage efficiency of the Cas13d effectors 46,73 , suggesting potential for future implementation. Furthermore, because they target RNA, next-generation Cas13-based systems may be capable of direct recognition of RNA, possibly at the single molecule level, without need for a prior reverse transcription (RT) and/or amplification step.…”

A widespread family of WYL-domain transcriptional regulators co-localises with diverse phage defence systems and islands

“…A handful of studies have begun to demonstrate that WYL-domain containing proteins regulate diverse processes in prokaryotes: Sll7009 from Synechocystis 6803 represses the CRISPR subtype I-D locus (28); DriD from Caulobacter crescentus activates expression of SOS-independent DNA damage response mediators (29); PIF1 helicase from Thermotoga elfii has a ssDNA-binding WYL-domain that couples ATPase activity to DNA unwinding (30); the RNA cleavage activity of a Type VI Cas13d protein from Ruminococcus is stimulated by a WYL-domain protein named WYL1 (31), which binds ssRNA with high affinity (32); and in Mycobacteria, PafBC is a transcriptional activator of DNA damage response genes (33). Most recently, WYL-domain proteins were found associated with phage defence islands within integrative conjugative elements of Vibrio cholerae (34).…”

Comparative Genomics and Physiological Investigation of a New Arthrospira/Limnospira Strain O9.13F Isolated from an Alkaline, Winter Freezing, Siberian Lake

“…Among them, we found 14 genes encoding hypothetical proteins, RNA-binding S4 domain-containing protein as well as two genes encoding four helix bundle proteins. Moreover, unique CDs encode proteins involved in the immunity system, N-6 DNA methylase, which is most similar with Methylase AflIII, the DUF433 domain-containing protein that might be a part of the putative toxin/antitoxin system ([ 96 ]) as well as the WYL domain-containing protein, which is a regulator of the Type VI-D CRISPR-Cas system [ 97 ] and thus might regulate the response to environmental stresses [ 98 ] ( Table 4 ).…”

“…Comparative genomics revealed no apparent characteristic genes that may be directly responsible for the adaptation of the Siberian strain to low temperatures of its habitat during the winter period. However, among unique CDs, we found those which encode proteins that might regulate the response to environmental stresses, such as methylase, the putative toxin/antitoxin system or a regulator of the CRISPR-Cas system [ 96 , 97 , 98 ]. What is more, strain O9.13F did not possess a different set of genes associated with the stress response than tropical strains.…”