image: Illustration of Cas9 binding to DNA. The top figure shows two nucleosomes surrounding a nucleosome-free stretch of DNA. Hypothetical PAM sites for Cas9 targets in the central region and the right-hand nucleosome are shown in red. The lower figure shows Cas9 (with sgRNA) bound to the central target, with the PAM and flanking DNA held deep in the protein cleft. The PAM in the nucleosome could not be accessed without dissociating the DNA from the histone core. DNA backbones are blue; the RNA backbone is teal; DNA and RNA bases are white, except for the PAM; histones are green; Cas9 is purple. view more
Credit: Image courtesy of Janet Iwasa (University of Utah, Salt Lake City).
Researchers report evidence in yeast cells that nucleosomes inhibit binding and cleavage by the genome-editing enzyme CRISPR-Cas9. Previous studies have revealed that the commonly used genome-editing enzyme Cas9, from Streptococcus pyogenes, balks at target DNA sequences assembled into nucleosomes, structures in which DNA is wound around protein spools for packaging into chromosomes. However, whether nucleosomes inhibit Cas9 binding and target DNA cleavage in living cells has been unclear. Using real-time analysis, Dana Carroll and colleagues report that Cas9 cleaves target DNA sequences in two gene-regulating stretches--HO and PHO5 promoters--of the yeast genome more efficiently when the targets are located in nucleosome-depleted rather than nucleosome-bound sites. Experimentally evicting nucleosomes through the insertion of binding sites for a nucleosome-displacing protein restored Cas9 target binding and cleavage to levels comparable to naturally nucleosome-depleted sites. Conversely, increasing nucleosome occupancy through mutation of binding sites for a gene switch at the HO locus reduced Cas9 cleavage, suggesting that nucleosomes inhibit Cas9 cleavage. In contrast, zinc finger nucleases, also common genome-editing tools, appeared largely indifferent to the presence of nucleosomes at target sites. Thus, the authors suggest, nucleosome position maps might help improve genome-editing efficiency in some applications, including human somatic therapies aimed at editing nondividing cells, in which nucleosome positions are relatively static, and base-editing efforts, in which Cas9 may need to sift through nucleosomes to edit target sequences without cleaving them.
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Article #18-10062: "Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo," by Robert Yarrington, Surbhi Verma, Shaina Schwartz, Jonathan Trautman, and Dana Carroll
MEDIA CONTACT: Dana Carroll, University of Utah, Salt Lake City, UT; tel: 801-581-5977; e-mail: dana@biochem.utah.edu
Journal
Proceedings of the National Academy of Sciences