Multiplexed Single-Molecule Experiments Reveal Nucleosome Invasion Dynamics of the Cas9 Genome Editor

J Am Chem Soc. 2021 Oct 13;143(40):16313-16319. doi: 10.1021/jacs.1c06195. Epub 2021 Oct 1.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Nucleosomes*


  • Nucleosomes