Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin.

Autor:	Strohkendl I; Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA. istrohkendl@utexas.edu rick_russell@cm.utexas.edu ilya@finkelsteinlab.org., Saifuddin FA; Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA.; Department of Cell and Tissue Biology, University of California, San Francisco, 513 Parnassus Ave, San Francisco, CA 94143, USA., Gibson BA; Department of Biophysics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA., Rosen MK; Department of Biophysics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA., Russell R; Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA. istrohkendl@utexas.edu rick_russell@cm.utexas.edu ilya@finkelsteinlab.org., Finkelstein IJ; Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA. istrohkendl@utexas.edu rick_russell@cm.utexas.edu ilya@finkelsteinlab.org.; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA.
Jazyk:	angličtina
Zdroj:	Science advances [Sci Adv] 2021 Mar 10; Vol. 7 (11). Date of Electronic Publication: 2021 Mar 10 (Print Publication: 2021).
DOI:	10.1126/sciadv.abd6030
Abstrakt:	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. (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)
Databáze:	MEDLINE
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