Multiplexed Single-Molecule Experiments Reveal Nucleosome Invasion Dynamics of the Cas9 Genome Editor
| Autor: | Carolin Anders, Kristina Makasheva, Louise C. Bryan, Martin Jinek, Sherin Panikulam, Beat Fierz |
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| Přispěvatelé: | University of Zurich, Fierz, Beat |
| Rok vydání: | 2021 |
| Předmět: |
binding
1303 Biochemistry 1503 Catalysis 610 Medicine & health 1600 General Chemistry 1505 Colloid and Surface Chemistry Computational biology dna Biochemistry Genome endonuclease Catalysis DNA sequencing interrogation 03 medical and health sciences chemistry.chemical_compound core particle 0302 clinical medicine Colloid and Surface Chemistry 10019 Department of Biochemistry Nucleosome CRISPR crispr 030304 developmental biology 0303 health sciences Nuclease biology Chemistry Cas9 Communication determinants General Chemistry Chromatin Nucleosomes kinetics biology.protein 570 Life sciences 030217 neurology & neurosurgery DNA |
| Zdroj: | Journal of the American Chemical Society JACS |
| DOI: | 10.5281/zenodo.7360318 |
| Popis: | Single-molecule measurements provide detailed mechanistic insights into molecular processes, for example in genome regulation where DNA access is controlled by nucleosomes and the chromatin machinery. However, real-time single-molecule observations of nuclear factors acting on defined chromatin substrates are challenging to perform quantitatively and reproducibly. Here we present XSCAN (multiplexed single-molecule detection of chromatin association), a method to parallelize single-molecule experiments by simultaneous imaging of a nucleosome library, where each nucleosome type carries an identifiable DNA sequence within its nucleosomal DNA. Parallel experiments are subsequently spatially decoded, via the detection of specific binding of dye-labeled DNA probes. We use this method to reveal how the Cas9 nuclease overcomes the nucleosome barrier when invading chromatinized DNA as a function of PAM position. |
| Databáze: | OpenAIRE |
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