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Type: Journal article
Title: Programmable DNA looping using engineered bivalent dCas9 complexes
Author: Hao, N.
Shearwin, K.
Dodd, I.
Citation: Nature Communications, 2017; 8(1):1-12
Publisher: Nature Publishing Group
Issue Date: 2017
ISSN: 2041-1723
Statement of
Nan Hao, Keith E. Shearwin, Ian B. Dodd
Abstract: DNA looping is a ubiquitous and critical feature of gene regulation. Although DNA looping can be efficiently detected, tools to readily manipulate DNA looping are limited. Here we develop CRISPR-based DNA looping reagents for creation of programmable DNA loops. Cleavage-defective Cas9 proteins of different specificity are linked by heterodimerization or translational fusion to create bivalent complexes able to link two separate DNA regions. After model-directed optimization, the reagents are validated using a quantitative DNA looping assay in E. coli. Looping efficiency is ~15% for a 4.7 kb loop, but is significantly improved by loop multiplexing with additional guides. Bivalent dCas9 complexes are also used to activate endogenous norVW genes by rewiring chromosomal DNA to bring distal enhancer elements to the gene promoters. Such reagents should allow manipulation of DNA looping in a variety of cell types, aiding understanding of endogenous loops and enabling creation of new regulatory connections.
Keywords: Escherichia coli; Endonucleases; Bacterial Proteins; DNA, Bacterial; Nucleic Acid Conformation; Enhancer Elements, Genetic; Promoter Regions, Genetic
Rights: © The Author(s) 2017, Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit licenses/by/4.0/.
RMID: 0030077814
DOI: 10.1038/s41467-017-01873-x
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Appears in Collections:Molecular and Biomedical Science publications

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