scholarly journals Inhibition of CRISPR-Cas12a DNA Targeting by Nucleosomes and Chromatin

2020 ◽  
Author(s):  
Isabel Strohkendl ◽  
Fatema A. Saifuddin ◽  
Bryan A. Gibson ◽  
Michael K. Rosen ◽  
Rick Russell ◽  
...  
Keyword(s):  
Dna Binding ◽  
Histone Modifications ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Eukaryotic Cells ◽  
Loop Formation ◽  
Core Particle ◽  
Dna Targeting ◽  
Dna Binding And Cleavage ◽  
Nucleosome Arrays

AbstractGenome engineering nucleases, including CRISPR-Cas12a, must access chromatinized DNA. Here, we investigate how Acidaminococcus sp. Cas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates DNA target accessibility to Cas12a. Nucleosome unwrapping determines the extent to which both steps of Cas12a binding–PAM recognition and R-loop formation–are inhibited by the nucleosome. Nucleosomes inhibit Cas12a binding even beyond the canonical core particle. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing a target-proximal nuclease-inactive Cas9 enhances DNA cleavage rates over 10-fold. Surprisingly, Cas12a readily cleaves DNA linking nucleosomes within chromatin-like phase separated nucleosome arrays—with DNA targeting reduced only ~4-fold. This work provides a mechanism for the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted regions of cells. We conclude that nucleosome wrapping restricts accessibility to CRISPR-Cas nucleases and anticipate that increasing nucleosome breathing dynamics will improve DNA binding and cleavage in eukaryotic cells.

scholarly journals Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin

2021 ◽  
Vol 7 (11) ◽  
pp. eabd6030
Author(s):  
Isabel Strohkendl ◽  
Fatema A. Saifuddin ◽  
Bryan A. Gibson ◽  
Michael K. Rosen ◽  
Rick Russell ◽  
...  
Keyword(s):  
Histone Modifications ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Eukaryotic Cells ◽  
Loop Formation ◽  
Chromatin Compaction ◽  
Dna Targeting ◽  
Genomic Regions ◽  
Dna Bendability ◽  
Nucleosome Arrays

Genome engineering nucleases must access chromatinized DNA. Here, we investigate how AsCas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates target accessibility to Cas12a and determines the extent to which both steps of binding—PAM recognition and R-loop formation—are inhibited by the nucleosome. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing target-proximal dCas9 enhances DNA cleavage rates over 10-fold. Unexpectedly, Cas12a readily cleaves internucleosomal linker DNA within chromatin-like, phase-separated nucleosome arrays. DNA targeting is reduced only ~5-fold due to neighboring nucleosomes and chromatin compaction. This work explains the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted genomic regions in cells. We conclude that nucleosome unwrapping regulates accessibility to CRISPR-Cas nucleases and propose that increasing nucleosome breathing dynamics will improve DNA targeting in eukaryotic cells.

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 Synthesis of novel calix[4]arene p-benzazole derivatives and investigation of their DNA binding and cleavage activities with molecular docking and experimental studies

RSC Advances ◽  
2020 ◽  
Vol 10 (63) ◽  
pp. 38695-38708
Author(s):  
Seyda Cigdem Ozkan ◽  
Fatma Aksakal ◽  
Aydan Yilmaz
Keyword(s):  
Molecular Docking ◽  
Dna Binding ◽  
Dna Cleavage ◽  
Experimental Studies ◽  
Binding Properties ◽  
Dna Binding And Cleavage

In this study, p-benzazole-derived calix[4]arene compounds with aromatic structures are synthesized and their DNA cleavage/binding properties are investigated.

Syntheses, crystal structures, DNA binding, DNA cleavage, molecular docking and DFT study of Cu(ii) complexes involving N2O4 donor azo Schiff base ligands

2018 ◽  
Vol 42 (1) ◽  
pp. 246-259 ◽  
Author(s):  
Saikat Banerjee ◽  
Pravat Ghorai ◽  
Paula Brandão ◽  
Dipanjan Ghosh ◽  
Sutanwi Bhuiya ◽  
...  
Keyword(s):  
Schiff Base ◽  
Molecular Docking ◽  
Dna Binding ◽  
Crystal Structures ◽  
Dna Cleavage ◽  
Dft Study ◽  
Schiff Base Ligands ◽  
Dna Binding And Cleavage

DNA binding and cleavage properties of three novel copper(ii) complexes involving azo Schiff base ligands have been studied.

Insights into the Mechanism of DNA Cleavage by Dynemicin A as Revealed by DNA-Binding and -Cleavage Studies of Synthetic Analogs

1995 ◽  
Vol 117 (28) ◽  
pp. 7574-7575 ◽  
Author(s):  
Andrew G. Myers ◽  
Scott B. Cohen ◽  
Norma J. Tom ◽  
David J. Madar ◽  
Mark E. Fraley
Keyword(s):  
Dna Binding ◽  
Dna Cleavage ◽  
Synthetic Analogs ◽  
Dna Binding And Cleavage

scholarly journals Kinetic basis for DNA target specificity of CRISPR-Cas12a

2018 ◽  
Author(s):  
Isabel Strohkendl ◽  
Fatema A. Saifuddin ◽  
James R. Rybarski ◽  
Ilya J. Finkelstein ◽  
Rick Russell
Keyword(s):  
Dna Cleavage ◽  
Target Sequence ◽  
Seed Region ◽  
Target Specificity ◽  
Loop Formation ◽  
Guide Rna ◽  
Dna Targeting ◽  
Cas9 Nuclease ◽  
Protospacer Adjacent Motif ◽  
Late Transition

SUMMARYClass II CRISPR-Cas nucleases are programmable via a single guide RNA, enabling genome editing applications in nearly all organisms. However, DNA cleavage at off-target sites that resemble the target sequence is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by Acidaminococcus sp Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically-separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands. Despite the functionally irreversible binding, Cas12a discriminates strongly against mismatches along most of the DNA target sequence, implying substantial reversibility during R-loop formation –a late transition state– and the absence of a ‘seed’ region. Our results provide a quantitative underpinning for the DNA cleavage patterns measured in vivo and observations of greater reported target specificity of Cas12a than the Cas9 nuclease.

scholarly journals Synthesis, spectral characterization, DNA interaction studies, anthelmintic and antimicrobial activity of transition metal complexes with 3-(2- hydroxybenzylideneamino)-2-methylquinazolin-4(3H)-one and 1,10- phenanthroline

2011 ◽  
Vol 1 (4) ◽  
pp. 127-138 ◽  
Keyword(s):  
Antimicrobial Activity ◽  
Dna Binding ◽  
Dna Cleavage ◽  
Thermal Studies ◽  
Dna Interaction ◽  
Molar Conductance ◽  
H Nmr ◽  
Elemental Analyses ◽  
Dna Binding And Cleavage

Mixed ligand complexes of cobalt(II) (1), copper(II) (2) and oxovanadium(IV) (3) with 3- (2-hydroxy benzylideneamino)-2-methylquinazolin-4(3H)-one and 1,10-phenanthroline have been synthesized and characterized by elemental analyses, IR, electronic, 1 H-NMR, mass spectra, molar conductance and thermal studies. The synthesized compounds were screened for their in vitro antimicrobial activity. The results show a significant increase in antimicrobial activity of the complexes compared to ligand. Antihelmintic activity of the compounds has been tested on earthworms and the enhanced activity was observed upon complexation. In addition, DNA binding and DNA cleavage studies for the newly prepared compounds were also studied. These studies indicate that the DNA binding and cleavage efficacy were increased in the complexes relative to the parental ligand.

Dinuclear Cd(ii), Mn(ii) and Cu(ii) complexes derived from (anthraquinone-1-diyl) benzoate: DNA binding and cleavage studies

RSC Advances ◽  
2014 ◽  
Vol 4 (87) ◽  
pp. 46639-46645 ◽  
Author(s):  
Lin Liu ◽  
Gong-Ming Zhang ◽  
Ru-Gang Zhu ◽  
Yong-Hui Liu ◽  
Hui-Meng Yao ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Cleavage ◽  
Dna Binding And Cleavage ◽  
Solvothermal Conditions

Three dinuclear Cd(ii), Mn(ii) and Cu(ii) complexes have been successfully synthesized under solvothermal conditions. Among them, only the Cu(ii) complex has the activity for DNA cleavage.

scholarly journals Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system

2019 ◽  
Author(s):  
Tyler S. Halpin-Healy ◽  
Sanne E. Klompe ◽  
Samuel H. Sternberg ◽  
Israel S. Fernández
Keyword(s):  
Genome Engineering ◽  
De Novo ◽  
Structural Basis ◽  
Functional Coupling ◽  
Type I ◽  
Loop Formation ◽  
Dna Targeting ◽  
Associated Proteins ◽  
Mechanistic Basis ◽  
Cascade Complex

AbstractBacteria have evolved adaptive immune systems encoded by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated (Cas) genes to maintain genomic integrity in the face of relentless assault from pathogens and mobile genetic elements [1–3]. Type I CRISPR-Cas systems canonically target foreign DNA for degradation via the joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas3 [4,5] but nuclease-deficient Type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons [6,7]. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion has remained elusive. Here we determined structures of a novel TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using single particle electron cryo-microscopy (cryo-EM), revealing the mechanistic basis of this functional coupling. The quality of the cryo-EM maps allowed for de novo modeling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3’ end of the crRNA. The natural Cas8-Cas5 fusion protein binds the 5’ crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer adjacent motif (PAM) recognition and R-loop formation. The present work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposon-associated proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome engineering applications.

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