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Functional annotation of native enhancers with a Cas9-histone demethylase fusion.

Nat. Methods. 2015; 
Kearns Nicola A,Pham Hannah,Tabak Barbara,Genga Ryan M,Silverstein Noah J,Garber Manuel,Maehr R
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Gene Synthesis A human codon–optimized, nuclease-inactive version of Nm Cas9 was gene synthesized with two SV40 nuclear localization signals and a triple Flag tag (GenScript Technologies) and cloned in-frame with either a BirA affinity tag, a KRAB repressor or LSD1 (for amino acid sequences, see Supplementar y Fig. Get A Quote

摘要

Understanding of mammalian enhancers is limited by the lack of a technology to rapidly and thoroughly test the cell type-specific function. Here, we use a nuclease-deficient Cas9 (dCas9)-histone demethylase fusion to functionally characterize previously described and new enhancer elements for their roles in the embryonic stem cell state. Further, we distinguish the mechanism of action of dCas9-LSD1 at enhancers from previous dCas9-effectors.

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