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dCas9 Fluorescence Loss Assays

The strength of dCas9 inhibition was measured in an Fluorescence Loss Assay. GFP, under the control of the lacUV5 promoter, is genomically inserted in an E. coli DH10ß strain. Three spacer were designed (Sp1 - 3) targeting either the -10 area of the lacUV5 promoter (Sp1) or the first 100 nucleotides of the GFP cds. A non-targeting Spacer (SpNT) was included as a control (See Larson et al., 2013, for design of spacers). All spacers were expressed via a single guide (sg)RNA expression cassette. Expression of dCas9 was IPTG inducible via the lacI/lacoperator system. Sp1 - 3 show clear reduction of GFP levels.

Figure1: Fluorescence Loss Assay. Amount of GFP fluorescence under the induction of dCas9 targeting GFP.

The expression of dCas9 is regulated by an IPTG inducible expression system. Sp1 - 3 represent three different spacers targeting GFP or the associated promoter. SpNT represents a non-targeting spacer acting as a control.

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