Programmable DNA looping using engineered bivalent dCas9 complexes. Issue 1 (December 2017)

• Record Type: Journal Article
• Authors: Hao, Nan; Shearwin, Keith; Dodd, Ian
• Is Part Of: Nature communications. Volume 8:Issue 1(2017)
• Page Start: 1
• Page End: 12
• Publication Date: 2017-12
• Subjects: Biology -- Periodicals; Physical sciences -- Periodicals; 505
• DOI: 10.1038/s41467-017-01873-x
• Languages: English
• ISSNs: 2041-1723

Abstract

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 inE. 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 endogenousnorVW 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. DNA loops are a ubiquitious feature of gene regulation across the kingdoms of life. Here the authors design a Cas9-based dimerization system for inducing DNA loops inE. coli, allowing activation and rewiring of gene expression.