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Heat-Triggered Remote Control of CRISPR-dCas9 for Tunable Transcriptional Modulation.

ACS Chem Biol. 2020; 
Gamboa L, Phung EV, Li H, Meyers JP, Hart AC, Miller IC, Kwong GA,,,,.
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Custom Vector Construction … The hU6 promoter and the activating guide scaffold (sgRNA 2.0)21 (IDT PAGE Ultramer) were simultaneously inserted into the pUC57 cloning vector (GenScript SD1176) by restriction enzyme digest using EcoRI, NsiI, and BamHI. The NsiI site was then removed using the Q5 … Get A Quote

摘要

CRISPR-associated proteins (Cas) are enabling powerful new approaches to control mammalian cell functions, yet the lack of spatially defined, noninvasive modalities limits their use as biological tools. Here, we integrate thermal gene switches with dCas9 complexes to confer remote control of gene activation and suppression with short pulses of heat. Using a thermal switch constructed from the heat shock protein A6 (HSPA6) locus, we show that a single heat pulse 3-5 °C above basal temperature is sufficient to trigger expression of dCas9 complexes. We demonstrate that dCas9 fused to the transcriptional activator VP64 is functional after heat activation, and, depending on the number of heat pulses, drives transcr... More

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