Title : Looping DNA
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Looping DNA
The formation of DNA loops is an integral mechanism in the control of gene expression, bringing various control regions of DNA, and their associated transcriptional factors and chromatin remodeling complexes, in proximity – even across long distances – to affect modulation of expression of the relevant genes. The ability to control such looping would be a new tool to target and modify specific gene expression for experimental and therapeutic benefit. A modification of the CRISPR system shows promise to effect such novel control mechanisms. 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.
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