The mechanisms of nuclease activation are explored by solving seven ternary cryo-electron mi-croscopy structures of wild-type Cas13d in complex with matched and mismatched targets and an active site loop in the HEPN domains that regulates substrate accessibility is identified.
Abstract
CRISPR-Cas13d is increasingly used for RNA knockdowns due to its programmability, but off-target RNA binding and cleavage of near-cognate RNAs hinder its broader adoption. Here, we explore the mechanisms of nuclease activation by solving seven ternary cryo-electron mi-croscopy structures of wild-type Cas13d in complex with matched and mismatched targets. These structures reveal a series of active, intermediate, and inactive states that illustrate a detailed activation mechanism. The crRNA undergoes dramatic conformational changes upon target RNA binding, with the helical-1 domain transitioning from an initially docked state with the N-terminal domain to an allosterically switched conformation that stabilizes the RNA duplex. Quantitative kinetics reveal that a single proximal mismatch preserves nanomolar binding affinity but completely abolishes nuclease activity by trapping Cas13d in an inactive state. We identify an active site loop in the HEPN domains that regulates substrate accessibility, with alanine scanning mutagenesis revealing both hypo- and hyperactivated variants. These findings establish the structural basis for Cas13d’s exquisite mismatch surveillance and provide a mechanistic framework for engineering RNA-targeting specificity and activity across HEPN nuclease family members.
It is shown that short RNAs can directly occupy the canonical crRNA-binding channel and trigger a catalytically competent trans cleavage state in the absence of PAM recognition or canonical R-loop formation.
I. Iwe, S. Singh, K. Guan et al.· medRxiv· 0 citations
It is shown that activation occurs only when overhang positioning creates an accessible protein–DNA interface, and a structural accessibility principle for LbuCas13a activation by noncontiguous DNA is defined.
Wei-Tao Wang, Yu-Han Chen, Ziyun Li et al.· Nucleic Acids Research· 0 citations
CRISPR-Cas12a is widely utilized for genome engineering and nucleic acid diagnostics, being distinguished by its indiscriminate single-stranded DNA (ssDNA) trans-cleavage activity triggered by its specific cis-target recognition. However, the precise kinetic coordination between these dual catalytic modes remains unclear because of methodological limitations, which preclude simultaneous monitoring of both activities. Here, we established a real-time, dual-wavelength fluorescence reporter system to dissect these dynamics utilizing phosphorothioate (PS) backbone modifications as chemical probes to interrogate enzyme turnover. We identified a functional decoupling and an asymmetric competitive mechanism strictly governed by “channel occupancy”. Specifically, we found that the PS modification of the cis-target abolished the trans-activity via a “product release gating” mechanism, where high-affinity product retention occluded the active site. Furthermore, we identified a critical length-dependent regulatory regime for ssDNA reporters, while short, noncleavable ligands (5-nt) acted as passive spectator molecules, and long analogues (30-nt) functioned as potent competitive inhibitors. The long ligands induced an irreversible “kinetic trap”, creating a nonproductive complex where the enzyme was permanently sequestered because of the lack of cleavage-mediated release. These findings demonstrate that the availability of the RuvC catalytic channel is determined not by induced fit binding but by the chemical cleavability of the occupant. This study establishes an integrated Cas12a-regulated kinetic model and systematically investigates the simultaneous effects of PS-modification on the cis- and trans-hydrolytic activities of Cas12a. The findings provide a theoretical framework and additional insights for developing more precise gene-editing tools and designing Cas12a-based in vitro diagnostic platforms.
The crystal structure of AcrIIA17 is presented and its mechanism of Staphylococcus aureus Cas9 (SauCas9) inhibition is elucidated, identifying AcrIIA17 as an Acr protein that targets the Cas9 BH domain and reveal the BH domain as a regulatory checkpoint in Cas9 activation.
G. Kim, Hyo Been Jin, Yong-Jun Kang et al.· iScience· 0 citations
Cas12a is highly accommodative toward noncanonical activation pathways to the extent of flipping its identity to be a DNA-guided RNA-targeting effector. A sequence engineering approach was used to systematically identify desirable guide DNA (gDNA) sequence motifs to achieve comparable RNA targeting efficiency as the canonical RNA-guided Cas12a with good selectivity down to single-nucleotide mismatch. Importantly, we introduced a split gDNA design concept with greater energetic differences arising from subtle nucleotide changes to probe the key spacer features for effective Cas12a-gDNA activation. Similar to the canonical RNA-guided activation pathway, Cas12a was found to engage actively in the "seed-like" scaffold-proximal region while the scaffold-distal region was largely hybridization-driven. We further evolved the split gDNA design to enhance the sequence selectivity by up to 21-fold compared to a single gDNA design and achieve single-nucleotide discrimination among representative let-7 family members. This study has established a gDNA sequence design framework to reprogram Cas12a as a precise RNA targeting platform.
RAPID (RNA/DNA Advanced chimeric, PAM-independent, Integrated Nicking, Diagnostics), a nick-tuned, PAM-duplex-mediated platform for PAM-independent RNA and DNA detection and applies it to detect single-point mutations in ssDNA and RNA substrates, a challenge for traditional Cas12 and Cas13 systems.
I. Iwe, Frank X. Liu, A. Corsano et al.· Nucleic Acids Research· 0 citations
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