The dCas9 is a Cas9 nuclease mutant. Mutations D10A and H840A in the RucC and HNH domains, respectively, abolish cleavage but do not impair DNA binding. The dCas9 provides a simple and robust technology for gene repression and activation, and can target almost any DNA sequence aided by the sgRNA. The ω subunit can activate transcription by recruiting the RNAP holoenzyme. The fusion between dCas9 and ω subunit can activate gene transcription downstream of the protospacer. The CRISPR interference inhibits transcription by sterically blocking the RNA polymerase(RNAP).
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===Results===
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<p>We got three kinds of transformants, each harboring (a)pResponse-RFP, (b)pResponse-RFP plus pActivator without sgRNA, (c)pResponse-RFP plus pActivator with sgRNA. Overnight cultures of different colonies were transferred and grow into early logarithmic stage. Then, we added tetracycline to culture (b) and (c) to a final concentration of 100ug/ml, and incubated them overnight at 30℃. Samples were prepared according to protocol and loaded to 96-well plate. Below is the data acquired from microplate reader, with excitation wavelength at 584nm and emission wavelength at 607nm. Florescence/OD600 indicates the intensity of mRFP.</p>
