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Programmable DNA looping using engineered bivalent dCas9 complexes
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DOI：10.1038/s41467-017-01873-x.png)
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摘要
En 中文
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 similar to 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.
Keyword:
RNA-GUIDED ENDONUCLEASE
CRISPR-CAS NUCLEASES
GENE-REGULATION
TARGET DNA
ESCHERICHIA-COLI
IN-VIVO
TRANSCRIPTION
RANGE
RECOGNITION
REPRESSION
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期刊
IF:
15.7
论文数:
9.6W
被引数:
115.3W
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