Open AccessMultiplexed Single-Molecule Experiments Reveal Nucleosome Invasion Dynamics of the Cas9 Genome Editor
Author(s) -
Kristina Makasheva,
Louise C. Bryan,
Carolin Anders,
Sherin Panikulam,
Martin Jínek,
Beat Fierz
Publication year - 2021
Publication title -
journal of the american chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.115
H-Index - 612
eISSN - 1520-5126
pISSN - 0002-7863
DOI - 10.1021/jacs.1c06195
Subject(s) - nucleosome , chromatin , chemistry , dna , biophysics , computational biology , nuclease , dna sequencing , linker dna , biology , biochemistry
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 (multiple x ed s ingle-molecule detection of c hromatin a ssociatio n ), 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.