scholarly journals Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

2019 ◽  
Author(s):  
Ikenna C Okafor ◽  
Digvijay Singh ◽  
Yanbo Wang ◽  
Minhee Jung ◽  
Haobo Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Engineering ◽  
Dna Unwinding ◽  
Dependent Manner ◽  
Guide Rnas ◽  
Single Molecule Fret ◽  
Wide Range ◽  
The Cost ◽  
Single Molecule Analysis ◽  
Target Activity

Abstract Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5′ end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5′ end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

scholarly journals Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

2019 ◽  
Author(s):  
Ikenna C. Okafor ◽  
Digvijay Singh ◽  
Yanbo Wang ◽  
Minhee Jung ◽  
Haobo Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Engineering ◽  
Dna Unwinding ◽  
Dependent Manner ◽  
Guide Rnas ◽  
Single Molecule Fret ◽  
Wide Range ◽  
The Cost ◽  
Single Molecule Analysis ◽  
Target Activity

ABSTRACTCas9 has made a wide range of genome engineering applications possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity but at the cost of the on-target activity. DNA unwinding is the primary checkpoint before cleavage by Cas9 and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in Cas9. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two guanines added were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5’ end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

scholarly journals Mechanisms of improved specificity of engineered Cas9s revealed by single molecule analysis

2017 ◽  
Author(s):  
Digvijay Singh ◽  
Yanbo Wang ◽  
John Mallon ◽  
Olivia Yang ◽  
Jingyi Fei ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Unwinding ◽  
Cleavage Reaction ◽  
Cleavage Rate ◽  
Guide Rna ◽  
Slowing Down ◽  
Rna Targets ◽  
Cleavage Reactions ◽  
Additional Base ◽  
Single Molecule Analysis

ABSTRACTIn microbes, CRISPR-Cas systems provide adaptive immunity against invading genetic elements. Cas9 in complex with a guide-RNA targets complementary DNA for cleavage and has been repurposed for wide-ranging biological applications. New Cas9s have been engineered (eCas9 and Cas9-HF1) to improve specificity, but how they help reduce off-target cleavage is not known. Here, we developed single molecule DNA unwinding assay to show that sequence mismatches affect cleavage reactions through rebalancing the internal unwinding/rewinding equilibrium. Increasing PAM-distal mismatches facilitate rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily upon mismatch detection. Intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. DNA interrogation experiments showed that engineered Cas9s require about one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity (1) by inhibiting stable DNA binding to partially matching sequences, (2) by making DNA unwinding more sensitive to mismatches, and (3) by slowing down intrinsic cleavage reaction.

Quantitative single molecule FRET efficiencies using TIRF microscopy

2015 ◽  
Vol 184 ◽  
pp. 131-142 ◽  
Author(s):  
Lasse L. Hildebrandt ◽  
Søren Preus ◽  
Victoria Birkedal
Keyword(s):  
Single Molecule ◽  
Structural Information ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Complexes ◽  
Distance Information ◽  
Single Molecule Level ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Intermolecular Distances

Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2–10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (http://www.isms.au.dk), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

scholarly journals Bulk and single-molecule analysis of a novel DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor

2019 ◽  
Author(s):  
O.J. Wilkinson ◽  
C. Carrasco ◽  
C. Aicart-Ramos ◽  
F. Moreno-Herrero ◽  
M.S. Dillingham
Keyword(s):  
Single Molecule ◽  
Nuclease Activity ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Dna Looping ◽  
Dna Unwinding ◽  
Ssdna Binding ◽  
Dna Replication And Repair ◽  
Essential Enzyme ◽  
Single Molecule Analysis

ABSTRACTDNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a novel bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5’ to 3’ ssDNA translocase and 5’ to 3’ helicase activity. Single molecule analysis reveals that Bad is a highly processive DNA motor capable of moving along DNA for distances of more than 4 kbp at a rate of ∼200 base pairs per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments also show that the enzyme loops DNA while translocating, which is an emerging feature of highly processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.

scholarly journals A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

2017 ◽  
Author(s):  
Yavuz S. Dagdas ◽  
Janice S. Chen ◽  
Samuel H. Sternberg ◽  
Jennifer A. Doudna ◽  
Ahmet Yildiz
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Divalent Cations ◽  
Guide Rna ◽  
Single Molecule Fret ◽  
Target Sites ◽  
Multiple Conformations ◽  
Dna Binding And Cleavage

AbstractThe Cas9 endonuclease is widely utilized for genome engineering applications by programming its single-guide RNA and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule FRET, we identified an intermediate state of S. pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. HNH docking requires divalent cations, but not strand scission, and this docked conformation persists following DNA cleavage. Sequence mismatches between the DNA target and guide RNA prevent transitions from the checkpoint intermediate to the active conformation, providing selective avoidance of DNA cleavage at stably bound off-target sites.

scholarly journals Pausing controls branching between productive and non-productive pathways during initial transcription

2017 ◽  
Author(s):  
David Dulin ◽  
David L. V. Bauer ◽  
Anssi M. Malinen ◽  
Jacob J. W. Bakermans ◽  
Martin Kaller ◽  
...  
Keyword(s):  
Single Molecule ◽  
Transcription Initiation ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Dependent Manner ◽  
Single Molecule Fret ◽  
Bacterial Rna ◽  
Tightly Coupled ◽  
Relationship Of ◽  
The Relationship

AbstractTranscription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. Recently, initial RNA synthesis by the bacterial RNA polymerase (RNAP) has been shown to be interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Here, we employed single-molecule FRET and biochemical analysis to disentangle the pausing-related pathways of bacterial initial transcription. We present further evidence that region σ3.2 constitutes a barrier after the initial transcribing complex synthesizes a 6-nt RNA (ITC6), halting transcription. We also show that the paused ITC6 state acts as a checkpoint that directs RNAP, in an NTP-dependent manner, to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway that blocks transcription initiation. Our results show that abortive RNA release and DNA unscrunching are not as tightly coupled as previously thought.

scholarly journals Integrating single-molecule FRET and biomolecular simulations to study diverse interactions between nucleic acids and proteins

2021 ◽  
Author(s):  
Joshua C. Sanders ◽  
Erik D. Holmstrom
Keyword(s):  
Nucleic Acids ◽  
Single Molecule ◽  
Dynamic Properties ◽  
Resonance Energy ◽  
Integrative Approach ◽  
Biological Macromolecules ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Biomolecular Simulations ◽  
Diverse Interactions

Abstract The conformations of biological macromolecules are intimately related to their cellular functions. Conveniently, the well-characterized dipole–dipole distance-dependence of Förster resonance energy transfer (FRET) makes it possible to measure and monitor the nanoscale spatial dimensions of these conformations using fluorescence spectroscopy. For this reason, FRET is often used in conjunction with single-molecule detection to study a wide range of conformationally dynamic biochemical processes. Written for those not yet familiar with the subject, this review aims to introduce biochemists to the methodology associated with single-molecule FRET, with a particular emphasis on how it can be combined with biomolecular simulations to study diverse interactions between nucleic acids and proteins. In the first section, we highlight several conceptual and practical considerations related to this integrative approach. In the second section, we review a few recent research efforts wherein various combinations of single-molecule FRET and biomolecular simulations were used to study the structural and dynamic properties of biochemical systems involving different types of nucleic acids (e.g., DNA and RNA) and proteins (e.g., folded and disordered).

scholarly journals Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps

2017 ◽  
Author(s):  
Luuk Loeff ◽  
Stan J.J. Brouns ◽  
Chirlmin Joo
Keyword(s):  
Single Molecule ◽  
Adaptive Immune System ◽  
Dna Unwinding ◽  
Type I ◽  
Base Pairs ◽  
Spatiotemporal Resolution ◽  
Adaptive Immune ◽  
Single Molecule Fret ◽  
Target Dna ◽  
Loaded Mechanism

CRISPR-Cas loci provide an RNA-guided adaptive immune system against invading genetic elements. Interference in type I systems relies on the RNA-guided surveillance complex Cascade for target DNA recognition and the trans-acting Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here we employed single-molecule FRET to probe the helicase activity with a high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling in the target DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of three base pairs, that each underlie three successive 1-nt unwinding events. Reeling is highly repetitive, compensating for inefficient nicking activity of the nuclease domain. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure well controlled degradation of target DNA only.

scholarly journals Probing the kinetic and thermodynamic consequences of the tetraloop/tetraloop receptor monovalent ion-binding site in P4–P6 RNA by smFRET

2015 ◽  
Vol 43 (2) ◽  
pp. 172-178 ◽  
Author(s):  
Namita Bisaria ◽  
Daniel Herschlag
Keyword(s):  
Single Molecule ◽  
Specific Binding ◽  
Specific Interaction ◽  
Monovalent Cations ◽  
Folding Kinetics ◽  
Rna Molecules ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Monovalent Ion

Structured RNA molecules play roles in central biological processes and understanding the basic forces and features that govern RNA folding kinetics and thermodynamics can help elucidate principles that underlie biological function. Here we investigate one such feature, the specific interaction of monovalent cations with a structured RNA, the P4–P6 domain of the Tetrahymena ribozyme. We employ single molecule FRET (smFRET) approaches as these allow determination of folding equilibrium and rate constants over a wide range of stabilities and thus allow direct comparisons without the need for extrapolation. These experiments provide additional evidence for specific binding of monovalent cations, Na+ and K+, to the RNA tetraloop–tetraloop receptor (TL–TLR) tertiary motif. These ions facilitate both folding and unfolding, consistent with an ability to help order the TLR for binding and further stabilize the tertiary contact subsequent to attainment of the folding transition state.

scholarly journals ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Su Hyung Park ◽  
Nalae Kang ◽  
Eunho Song ◽  
Minwoo Wie ◽  
Eun A. Lee ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Cultured Cells ◽  
Replication Stress ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Single Molecule Fret ◽  
Hydroxyurea Treatment ◽  
Fork Regression

AbstractMaintaining stability of replication forks is important for genomic integrity. However, it is not clear how replisome proteins contribute to fork stability under replication stress. Here, we report that ATAD5, a PCNA unloader, plays multiple functions at stalled forks including promoting its restart. ATAD5 depletion increases genomic instability upon hydroxyurea treatment in cultured cells and mice. ATAD5 recruits RAD51 to stalled forks in an ATR kinase-dependent manner by hydroxyurea-enhanced protein-protein interactions and timely removes PCNA from stalled forks for RAD51 recruitment. Consistent with the role of RAD51 in fork regression, ATAD5 depletion inhibits slowdown of fork progression and native 5-bromo-2ʹ-deoxyuridine signal induced by hydroxyurea. Single-molecule FRET showed that PCNA itself acts as a mechanical barrier to fork regression. Consequently, DNA breaks required for fork restart are reduced by ATAD5 depletion. Collectively, our results suggest an important role of ATAD5 in maintaining genome integrity during replication stress.

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