scholarly journals A gate and clamp regulate sequential DNA strand cleavage by CRISPR-Cas12a

2021 ◽  
Author(s):  
Mohsin M Naqvi ◽  
Laura Lee ◽  
Oscar E Torres Montaguth ◽  
Mark Szczelkun
Keyword(s):  
Dna Recognition ◽  
Magnetic Tweezers ◽  
Dna Unwinding ◽  
Nanopore Sequencing ◽  
Loop Formation ◽  
Target Strand ◽  
Diagnostic Applications ◽  
Dna Strand ◽  
Domain Motions ◽  
Resistant State

CRISPR-Cas12a has been widely used for genome editing and diagnostic applications, yet it is not fully understood how RNA-guided DNA recognition activates the sequential cleavage of the non-target strand (NTS) followed by the target strand (TS). Here we used singlemolecule magnetic tweezers microscopy, ensemble gel-based assays and nanopore sequencing to explore the coupling of DNA unwinding and cleavage. In addition to dynamic R-loop formation, we also directly observed transient dsDNA unwinding downstream of the 20 bp DNA:RNA hybrid and, following NTS cleavage and prior to TS cleavage, formation of a hyperstable "clamped" Cas12a-DNA intermediate resistant to DNA twisting. Alanine substitution of a conserved aromatic amino acid "gate" in the REC2 domain that normally caps the heteroduplex produced more frequent and extended downstream DNA breathing, a longer-lived twist-resistant state, and a 16-fold faster rate of TS cleavage. We suggest that both breathing and clamping events, regulated by the gate and by NTS cleavage, deliver the unwound TS to the RuvC nuclease and result from previously described REC2 and NUC domain motions.

scholarly journals The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 169 ◽  
Author(s):  
Kara van Aelst ◽  
Carlos Martínez-Santiago ◽  
Stephen Cross ◽  
Mark Szczelkun
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Streptococcus Thermophilus ◽  
Dna Supercoiling ◽  
Magnetic Tweezers ◽  
Loop Formation ◽  
Formation Kinetics ◽  
Target Strand ◽  
Dna Strand ◽  
Type V

Here we explored the mechanism of R-loop formation and DNA cleavage by type V CRISPR Cas12a (formerly known as Cpf1). We first used a single-molecule magnetic tweezers (MT) assay to show that R-loop formation by Lachnospiraceae bacterium ND2006 Cas12a is significantly enhanced by negative DNA supercoiling, as observed previously with Streptococcus thermophilus DGCC7710 CRISPR3 Cas9. Consistent with the MT data, the apparent rate of cleavage of supercoiled plasmid DNA was observed to be >50-fold faster than the apparent rates for linear DNA or nicked circular DNA because of topology-dependent differences in R-loop formation kinetics. Taking the differences into account, the cleavage data for all substrates can be fitted with the same apparent rate constants for the two strand-cleavage steps, with the first event >15-fold faster than the second. By independently following the ensemble cleavage of the non-target strand (NTS) and target strand (TS), we could show that the faster rate is due to NTS cleavage, the slower rate due to TS cleavage, as expected from previous studies.

scholarly journals Mechanism of R-loop formation and conformational activation of Cas9

2021 ◽  
Author(s):  
Martin Pacesa ◽  
Martin Jinek
Keyword(s):  
Dna Cleavage ◽  
Structural Basis ◽  
Seed Region ◽  
Loop Formation ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Strand ◽  
Target Dna ◽  
Dna Strand ◽  
Dna Substrate

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications. Despite extensive studies, the precise mechanism of target DNA binding and on-/off-target discrimination remains incompletely understood. Here we report cryo-EM structures of intermediate binding states of Streptococcus pyogenes Cas9 that reveal domain rearrangements induced by R-loop propagation and PAM-distal duplex positioning. At early stages of binding, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the PAM-distal duplex of the DNA substrate. Target hybridisation past the seed region positions the guide-target heteroduplex into the central binding channel and results in a conformational rearrangement of the REC lobe. Extension of the R-loop to 16 base pairs triggers the relocation of the HNH domain towards the target DNA strand in a catalytically incompetent conformation. The structures indicate that incomplete target strand pairing fails to induce the conformational displacements necessary for nuclease domain activation. Our results establish a structural basis for target DNA-dependent activation of Cas9 that advances our understanding of its off-target activity and will facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

scholarly journals Probing the stability of the SpCas9-DNA complex after cleavage

2021 ◽  
Author(s):  
Pierre Aldag ◽  
Fabian Welzel ◽  
Leonhard Jakob ◽  
Andreas Schmidbauer ◽  
Marius Rutkauskas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
Slowing Down ◽  
Target Strand ◽  
Dna Strands ◽  
Dna Strand ◽  
The Stability ◽  
The Individual ◽  
Dna Complex

CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 as well as its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different S. pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around the DNA with friction. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress. After target-strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability compared to the pre-cleavage state. This suggests that Cas9 has evolved to remain tightly bound to its cut target.

scholarly journals Mechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bo Zhang ◽  
Diyin Luo ◽  
Yu Li ◽  
Vanja Perčulija ◽  
Jing Chen ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Binary Complex ◽  
Loop Formation ◽  
Dna Duplex ◽  
Target Dna ◽  
Before And After ◽  
Dna Strand ◽  
Type V ◽  
Functionally Diverse

AbstractCas12i is a newly identified member of the functionally diverse type V CRISPR-Cas effectors. Although Cas12i has the potential to serve as genome-editing tool, its structural and functional characteristics need to be investigated in more detail before effective application. Here we report the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage to elucidate the mechanisms underlying target DNA duplex unwinding, R-loop formation and cis cleavage. The structure of the R-loop complex after target DNA cleavage also provides information regarding trans cleavage. Besides, we report a crystal structure of the Cas12i1 binary complex interacting with a pseudo target oligonucleotide, which mimics target interrogation. Upon target DNA duplex binding, the Cas12i1 PAM-interacting cleft undergoes a remarkable open-to-closed adjustment. Notably, a zipper motif in the Helical-I domain facilitates unzipping of the target DNA duplex. Formation of the 19-bp crRNA-target DNA strand heteroduplex in the R-loop complexes triggers a conformational rearrangement and unleashes the DNase activity. This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool.

scholarly journals Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

2021 ◽  
Author(s):  
Kazuto Yoshimi ◽  
Kohei TAKESHITA ◽  
Noriyuki Kodera ◽  
Satomi Shibumura ◽  
Yuko Yamauchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Speed ◽  
Dna Helicase ◽  
Type I ◽  
Loop Formation ◽  
Force Microscopy ◽  
Atomic Force ◽  
Target Strand ◽  
Target Dna

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific ssDNA cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target dsDNA substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

A Hexameric Helicase Encircles One DNA Strand and Excludes the Other during DNA Unwinding†

Biochemistry ◽  
1997 ◽  
Vol 36 (46) ◽  
pp. 14080-14087 ◽  
Author(s):  
Kevin J. Hacker ◽  
Kenneth A. Johnson
Keyword(s):  
The Other ◽  
Dna Unwinding ◽  
Dna Strand

scholarly journals DNA Unwinding Mechanism of a RecQ Helicase from Arabidopsis Thaliana Investigated with Magnetic Tweezers

2011 ◽  
Vol 100 (3) ◽  
pp. 23a
Author(s):  
Daniel Klaue ◽  
Daniela Kobbe ◽  
Holger Puchta ◽  
Ralf Seidel
Keyword(s):  
Arabidopsis Thaliana ◽  
Magnetic Tweezers ◽  
Dna Unwinding ◽  
Recq Helicase

scholarly journals Interaction of HelQ helicase with RPA modulates RPA-DNA binding and stimulates HelQ to unwind DNA through a protein roadblock

2019 ◽  
Author(s):  
Sarah J. Northall ◽  
Tabitha Jenkins ◽  
Denis Ptchelkine ◽  
Vincenzo Taresco ◽  
Christopher D. O. Cooper ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Unwinding ◽  
Abasic Site ◽  
Domain Interaction ◽  
Dna Helicases ◽  
Intrinsically Disordered ◽  
Catalytically Active ◽  
Dna Strands ◽  
Dna Strand ◽  
Protein Barrier

ABSTRACTCells reactivate compromised DNA replication forks using enzymes that include DNA helicases for separating DNA strands and remodelling protein-DNA complexes. HelQ helicase promotes replication-coupled DNA repair in mammals in a network of interactions with other proteins. We report newly identified HelQ helicase activities, when acting alone and when interacting with RPA. HelQ helicase was strongly inhibited by a DNA-protein barrier (BamHIE111A), and by an abasic site in the translocating DNA strand. Interaction of HelQ with RPA activated DNA unwinding through the protein barrier, but not through the abasic site. Activation was lost when RPA was replaced with bacterial SSB or DNA binding-defective RPA, RPAARO1. We observed stable HelQ-RPA-DNA ternary complex formation, and present evidence that an intrinsically disordered N-terminal region of HelQ (N-HelQ) interacts with RPA, destabilising RPA-DNA binding. Additionally, SEC-MALS showed that HelQ multimers are converted into catalytically active dimers when ATP-Mg2+ is bound. HelQ and RPA are proposed to jointly promote replication fork recovery by helicase-catalysed displacement of DNA-bound proteins, after HelQ gains access to ssDNA through its N-terminal domain interaction with RPA.

scholarly journals Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

2019 ◽  
Author(s):  
Hazal B. Kose ◽  
Sherry Xie ◽  
George Cameron ◽  
Melania S. Strycharska ◽  
Hasan Yardimci
Keyword(s):  
Single Molecule ◽  
Replication Fork ◽  
Double Helix ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
Replicative Helicase ◽  
Double Stranded Dna ◽  
Order Of Magnitude ◽  
Dna Strand ◽  
Dna Double Helix

AbstractThe DNA double helix is unwound by the Cdc45/Mcm2-7/GINS (CMG) complex at the eukaryotic replication fork. While isolated CMG unwinds duplex DNA very slowly, its fork unwinding rate is stimulated by an order of magnitude by single-stranded DNA binding protein, RPA. However, the molecular mechanism by which RPA enhances CMG helicase activity remained elusive. Here, we demonstrate that engagement of CMG with parental double-stranded DNA (dsDNA) at the replication fork impairs its helicase activity, explaining the slow DNA unwinding by isolated CMG. Using single-molecule and ensemble biochemistry, we show that binding of RPA to the excluded DNA strand prevents duplex engagement by the helicase and speeds up CMG-mediated DNA unwinding. When stalled due to dsDNA interaction, DNA rezipping-induced helicase backtracking re-establishes productive helicase-fork engagement underscoring the significance of plasticity in helicase action. Together, our results elucidate the dynamics of CMG at the replication fork and reveal how other replisome components can mediate proper DNA engagement by the replicative helicase to achieve efficient fork progression.

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