RNA-triggered CRISPR-Cas12a2 Preferentially and Cooperatively Cleaves Collateral DNA

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Янв. 5, 2025

Abstract CRISPR-Cas systems often rely on collateral cleavage activities against nucleic-acid substrates to combat recognized mobile genetic elements. Of the associated RNA-guided Cas effector nucleases, Cas12a2 stands out as only known example exhibiting RNA-triggered of three distinct substrates: single-stranded (ss)RNA, ssDNA, and double-stranded (ds)DNA. However, little is about underlying mechanisms cleavage, including which these dominates during Cas12a2-mediated immune response. Here, we show that cleaves DNA over RNA substrates, even when are more abundant. This preference relies a positive cooperativity mechanism requires four “aromatic clamp” residues stabilize unwound distorted dsDNA in RuvC nuclease active site. Leveraging for DNA, demonstrate RNA-activated can cleave ssDNA probe presence high concentrations non-target RNA, while RNA-targeting Cas13a cannot. work thus reveals how decides amongst different with immediate implications understanding Cas12a2-based immunity, improving molecular diagnostics, laying mechnastic foundation future technologies.

Язык: Английский

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Helical transition of the bridge helix of Cas12a is an allosteric regulator of R-loop formation and RuvC activation

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Янв. 10, 2025

ABSTRACT CRISPR-Cas12a is widely used for genome editing and biomarker detection since it can create targeted double-stranded DNA breaks promote non-specific cleavage after identifying specific DNA. To mitigate the off-target of Cas12a, we previously developed a Francisella novicida Cas12a variant (FnoCas12a KD2P ) by introducing double proline substitutions (K969P/D970P) in conserved helix called bridge (BH). In this work, cryogenic electron microscopy (cryoEM) to understand molecular mechanisms BH- mediated activation Cas12a. We captured five structures FnoCas12a at different states conformational activation. Comparison with wild-type WT unravels mechanism where BH acts as trigger that allosterically activates REC lobe movements tracking number base pairs growing RNA-DNA hybrid undergo loop-to-helical transition bending latch onto hybrid. The coupled reported loop-to-helix “lid”, essential opening RuvC endonuclease, through direct interactions residues lid. also observe structural details cooperativity “helix-1” activation, proposed interaction. Overall, our study enables development high-fidelity Cas9 variants BH-modifications.

Язык: Английский

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Analysis of metal-dependent DNA nicking activities by Cas endonucleases

Methods in enzymology on CD-ROM/Methods in enzymology, Год журнала: 2025, Номер unknown, С. 117 - 142

Опубликована: Янв. 1, 2025

Язык: Английский

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Structural basis for target DNA cleavage and guide RNA processing by CRISPR-Casλ2

Research Square (Research Square), Год журнала: 2025, Номер unknown

Опубликована: Март 19, 2025

RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse effectors, CRISPR-Casλ, a recently identified miniature type V effector encoded in phage genomes, has emerged promising candidate for genome editing due to its nuclease activity mammalian and plant cells. However, detailed molecular mechanisms of Casλ family enzymes remain poorly understood. In this study, we report identification biochemical structural characterizations CRISPR-Casλ2. The cryo-electron microscopy structures Casλ2 five different functional states unveiled dynamic domain rearrangements during activation. revealed that, unlike other REC2 directly interacts with substrate DNA within RuvC active site facilitate target cleavage. Our analyses indicated that processes precursor crRNA mature using through unique ruler mechanism, which defines spacer length crRNA. Furthermore, comparisons Casλ1 CasΦ highlighted diversity conservation phage-encoded enzymes. Collectively, our findings augment mechanistic understanding establish framework rational engineering CRISPR-Casλ-based genome-editing platform.

Язык: Английский

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A self-actuated CRISPR/Cas12a feedback amplification platform for ultrasensitive detection of exosome

Microchemical Journal, Год журнала: 2025, Номер unknown, С. 113477 - 113477

Опубликована: Март 1, 2025

Язык: Английский

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Directed evolution expands CRISPR-Cas12a genome editing capacity

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Март 26, 2025

CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture and human therapeutics. However, their dependence on a 5'-TTTV-3' protospacer-adjacent motif (PAM) next to DNA target sequences restricts Cas12a's gene targeting capability only ∼1% of typical genome. To mitigate this constraint, we used bacterial-based directed evolution assay combined rational engineering identify variants Lachnospiraceae bacterium Cas12a (LbCas12a) expanded PAM recognition. The resulting use range non-canonical PAMs while retaining recognition the canonical PAM. In particular, biochemical cell-based assays show that variant Flex-Cas12a utilizes 5'-NYHV-3' expand sites ∼25% With enhanced versatility, unlocks access previously inaccessible genomic loci, providing new opportunities for both therapeutic agricultural genome engineering.

Язык: Английский

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Unveiling Cas8 dynamics and regulation within a transposon-encoded Cascade–TniQ complex

Proceedings of the National Academy of Sciences, Год журнала: 2025, Номер 122(14)

Опубликована: Апрель 2, 2025

The Vibrio cholerae Cascade–TniQ complex unveiled a new paradigm in biology, demonstrating that CRISPR-associated proteins can direct DNA transposition. Despite the tremendous potential of “knocking-in” genes at desired sites, mechanisms underlying binding and transposition remain elusive. In this system, conformational change Cas8 protein is essential for binding, yet how it occurs unclear. Here, structural modeling free energy simulations reconstruct helical bundle reveal an open–closed key complex’s function. We show when binds RNA, changes conformation mediated by interaction with Cas7.1 protein. This promotes bundle’s transition toward open state, priming binding. As target guide opening becomes more favorable, exposing positively charged residues facilitating their DNA, which ultimately leads DNA-binding process to completion. These outcomes provide dynamic representation critical one largest CRISPR systems illustrate its role steps biophysical function, advancing our understanding nucleic acid mechanisms.

Язык: Английский

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Nonequilibrium hybridization-driven CRISPR/Cas adapter with extended energetic penalty for discrimination of single-nucleotide variants

Nucleic Acids Research, Год журнала: 2025, Номер 53(7)

Опубликована: Апрель 2, 2025

Accurate identification of single-nucleotide variants (SNVs) is critical in clinical diagnostics but remains challenging due to subtle free energy variations, particularly for hard-to-detect SNVs such as wobble base pairs and those high guanine-cytosine (GC) regions. Here we report a high-energetic-penalty SNV detection (HEPSD) platform that redesigns the hybridization regions CRISPR RNA (crRNA) CRISPR/Cas12a system. This system employs binary crRNA architecture design enables activation cleavage activity Cas12a while amplifying energetic penalty mismatches through nonequilibrium hybridization-driven regulation. Consequently, entire targeting region CRISPR/Cas exhibits marked preference mutations genomic DNA, preventing false induced by sequences containing single mismatched nucleotide. Moreover, HEPSD exceptional differentiation performance including at extreme GC contents. As proof principle, profiling BRAF V600E EGFR L858R tumor down 0.01% variant allele frequency was achieved, enabling accurate discrimination 132 sample pairs, which showed consistency with quantitative polymerase chain reaction-based techniques next-generation sequencing. The proven effectiveness this showcases its potential molecular expands fundamental scope hybridization-based protocols.

Язык: Английский

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Graph theory approaches for molecular dynamics simulations

Quarterly Reviews of Biophysics, Год журнала: 2024, Номер 57

Опубликована: Янв. 1, 2024

Graph theory, a branch of mathematics that focuses on the study graphs (networks nodes and edges), provides robust framework for analysing structural functional properties biomolecules. By leveraging molecular dynamics (MD) simulations, atoms or groups can be represented as nodes, while their dynamic interactions are depicted edges. This network-based approach facilitates characterization such connectivity, centrality, modularity, which essential understanding behaviour systems. review details application development graph theory-based models in studying biomolecular We introduce key concepts theory demonstrate practical applications, illustrating how innovative approaches employed to design systems with enhanced functionality. Specifically, we explore integration theoretical methods MD simulations gain deeper insights into complex biological phenomena, allosteric regulation, conformational dynamics, catalytic functions. Ultimately, has proven powerful tool field offering valuable properties, establishes foundation using engineering, highlighting its potential transform drive advancements manipulation

Язык: Английский

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Enhanced trans-cleavage activity using CRISPR-Cas12a variant designed to reduce steric inhibition by cis-cleavage products

Biosensors and Bioelectronics, Год журнала: 2024, Номер 267, С. 116859 - 116859

Опубликована: Окт. 16, 2024

Язык: Английский

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