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.
Abstract
Abstract CRISPR–Cas13a is mainly known as an RNA-guided RNA endonuclease. Recent studies show that Leptotrichia buccalis Cas13a (LbuCas13a) can interact with DNA substrates too, without PAM or PFS constraints, but current understanding of DNA-mediated activation is largely based on continuous target strands. Here, we define a structural accessibility principle for LbuCas13a activation by noncontiguous DNA. We show that activation occurs only when overhang positioning creates an accessible protein–DNA interface. Outer overhangs near the crRNA repeat-adjacent side restore strong trans-cleavage activity by stabilizing key LbuCas13a–DNA contacts, whereas distal outer overhangs support only weak activation. In contrast, inner overhangs cause steric mismatch, destabilize the complex, and block formation of an active conformation. Molecular modeling and molecular dynamics simulations support this structure-dependent rule. Noncontiguous DNA also broadens the single-nucleotide discrimination window of LbuCas13a and enables accurate IDH1 R132H detection in glioma tissues. We further develop a one-step APE1-activated CRISPR–LbuCas13a reaction (ACROSS) for sensitive APE1 detection. Because activated LbuCas13a cleaves RNA reporters but not DNA-triggering products, ACROSS preserves the activating structure and supports stable signaling in vitro, in live cells, and in breast cancer serum samples.
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.
Chia-Wei Chou, Selma Sinan, Hung-Che Kuo et al.· bioRxiv· 0 citations
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
The CRISPR/Cas12a system is widely used in nucleic-acid diagnostics because Cas12a couples guide-directed recognition of a cis-target with indiscriminate collateral trans-cleavage of ssDNA. Controlling these activities through DNA structure can tune diagnostic signals. G-quadruplexes (G4s) are strong structural regulators, yet their ability to modulate Cas12a activation and collateral cleavage remains undefined. Here we focused on the G4 scaffold (TGGG)n and tested it with LbCas12a and AsCas12a by real-time cleavage assays, circular dichroism, FRET and denaturing PAGE. In the cis position, compact (TGGG)n G4s activated Cas12a; for the most stable (TGGG)5, kcat/KM was 5.9×104 and 8.9×104 M−1 s−1 for LbCas12a and AsCas12a, respectively. K+ reduced cis-activation rates by approximately 10-fold, depending on scaffold and temperature. FRET and denaturing PAGE showed that productive cis-recognition involves G4 unfolding followed by target-strand cleavage. In the trans position, compact G4s fully resisted collateral reporter cleavage. Core disruption and G-rich non-G4 controls restored reporter cleavage, showing that resistance depends on G4 architecture rather than guanine content. These data define a compact G4 scaffold that remains functionally trans-resistant while retaining guide-dependent cis-target competence. Thus, compact G4 folding provides a programmable structural mechanism for separating Cas12a activation from reporter cleavage, opening a route to signal-gated diagnostic designs. Graphical abstract
I. Safenkova, Maria V. Kamionskaya, A. V. Samokhvalov et al.· bioRxiv· 0 citations
Synthetic riboswitches provide protein-independent, modular control of gene expression, yet selecting aptamers that reliably couple ligand binding to regulatory switching remains challenging. Here, we identify and mechanistically characterise G12, a doxycycline-binding aptamer with remarkably high regulatory performance in yeast and human cells. We provide evidence that RNA Capture-SELEX efficiently enriches aptamers with ligand-responsive conformational switching. We compared conventional SELEX and RNA Capture-SELEX using the same starting library followed by NGS analysis and in vivo screening, which led to the identification of G12. G12 binds doxycycline with low-nanomolar affinity and strict discrimination against close derivatives, thus enabling high-dynamic-range riboswitch control of translation in yeast and splicing in human cells. Single-molecule force spectroscopy with optical tweezers revealed that doxycycline stabilises a folding intermediate independent of the closing stem P1, which primarily acts as a scaffold for correct aptamer folding. Mutational analysis and chemical probing identified tertiary contacts between loops L2 and L3 in this intermediate state. Stopped-flow fluorescence spectroscopy further supported a two-step binding mechanism consistent with efficient regulatory switching. Together, these findings deepen our understanding of regulatory aptamer selection and function and expand the synthetic biology toolbox with a high-performance doxycycline-responsive riboswitch. Synthetic riboswitches provide protein-independent, modular control of gene expression, yet selecting aptamers that reliably couple ligand binding to regulatory switching remains challenging. In this study, the authors identified a regulatory RNA aptamer binding doxycycline for the engineering of synthetic riboswitches.
J. Hoetzel, A. Walbrun, M. Schäfer et al.· Nature Communications· 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.
