Makasheva, KristinaBryan, Louise C.Anders, CarolinPanikulam, SherinJinek, MartinFierz, Beat2021-11-202021-11-202021-11-202021-10-1310.1021/jacs.1c06195https://infoscience.epfl.ch/handle/20.500.14299/183082WOS:000709467900001Single-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 dyelabeled 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.Chemistry, MultidisciplinaryChemistrycore particlednabindingendonucleasecrisprinterrogationdeterminantskineticstext::journal::journal article::research article