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Illustration of the binding of Cas9 to DNA. The upper figure shows two nucleosomes surrounding a nucleosome-free segment of DNA. The hypothetical PAM sites for the Cas9 targets in the central region and the right nucleosome are indicated in red. The lower figure shows Cas9 (with sgRNA) bound to the central target, with MAP and flanking DNA held deep in the slit of the protein. PAM in the nucleosome could not be accessed without dissociating the histone core DNA. The DNA skeletons are blue; the skeleton of RNA is teal; The DNA and RNA bases are white, with the exception of PAM; the histones are green; Cas9 is purple. Credit: Janet Iwasa (University of Utah, Salt Lake City).
A team of researchers at the University of Utah discovered that nucleosomes can inhibit the efficiency of CRISPR / Cas9 cleavage. In their article published in Proceedings of the National Academy of Sciences, the group describes the test of gene editing technique on yeast samples and what they found.
The CRISPR-Cas 9 gene editing technique uses the guide RNA to find and extract DNA segments. But what happens when the targeted segment is part of a nucleosome? Previous research has suggested that in such cases the effectiveness of cleavage would probably be compromised. In this new effort, the researchers performed an in vivo test of such cases and found that the results of previous research were correct – using CRISPR-Cas 9 on nucleosomes might not work very well.
The DNA strands are tiny, but really long – about six feet long when they are stretched. Because of this, the cells have mechanisms for packing the DNA into a cell nucleus. This mechanism involves winding the strands into clusters around a given protein. Such rolled clusters are called nucleosomes. Logic suggests that a technique for modifying a strand of DNA might encounter difficulties because of accessibility issues. Other researchers have considered the possibility of such problems, but have studied them in test tubes or have done their work on known strands that are not part of the nucleosomes. In this new effort, the researchers sought to know once and for all whether CRISPR-Cas9 would work just as well for editing strands that are part of nucleosomes in living tissues as for strands that are not.
The work involved an edition of the CRISPR-Cas9 gene using various guide RNAs in live yeast, which allowed different targets to be modified. Some of the targets were in the nucleosomes while others were not.
The researchers report that cleavage efficiency was much lower in nucleosomes than in non-nucleosomal areas. But they also found something else: when they test the gene editing technique called Zinc Fingers in the same way, they found no difference. The group suggests that in the future, gene editing efforts should begin with nucleosomal position maps to improve efficiency.
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More information:
Robert M. Yarrington et al. Nucleosomes inhibit CRISPR-Cas9 target cleavage in vivo, Proceedings of the National Academy of Sciences (2018). DOI: 10.1073 / pnas.1810062115
Abstract
The genome edition with CRISPR-Cas nucleases has been successfully applied to a wide range of cells and organisms. There is however a considerable variation in cleavage efficiency and results for different genomic targets, even within the same cell type. Part of this variability is probably due to the inherent quality of the interaction between the guide RNA and the target sequence, but some may also reflect the relative accessibility of the target. We investigated the influence of chromatin structure, particularly the presence or absence of nucleosomes, on cleavage by the Cas9 protein of Streptococcus pyogenes. At multiple target sequences in two promoters of the yeast genome, we find that cleavage of Cas9 is strongly inhibited when the DNA target is in a nucleosome. This inhibition is relieved when the nucleosomes are depleted. Remarkably, the same is not true for zinc finger nucleases (ZFNs), which cleave on sites occupied by both nucleosomes and nucleosome-depleted sites. These results have implications for the selection of specific targets for genome editing, both in research as well as in clinical applications and other practical applications.
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