Please use this identifier to cite or link to this item:
https://hdl.handle.net/2440/110817
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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 2041-1723 |
Statement of Responsibility: | 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 CRISPR-Associated Protein 9 |
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 http://creativecommons.org/ licenses/by/4.0/. |
DOI: | 10.1038/s41467-017-01873-x |
Grant ID: | http://purl.org/au-research/grants/nhmrc/1100651 http://purl.org/au-research/grants/arc/DE150100091 http://purl.org/au-research/grants/arc/DP160101450 |
Published version: | http://dx.doi.org/10.1038/s41467-017-01873-x |
Appears in Collections: | Aurora harvest 8 Molecular and Biomedical Science publications |
Files in This Item:
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hdl_110817.pdf | Published Version | 1.14 MB | Adobe PDF | View/Open |
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