Fig. 3.

The ChaCha system enables activation of individual and of multiple genes. a Induction of endogenous IL2 and IFN-γ expression and secretion using the hM3D-CRISPR ChaCha system measured by ELISA. Gray, activation using free dCas9-VPR with a targeting (sgIL2 or sjpgNG) or non-targeting sgRNA (sgGAL4). Blue, activation of IL2 or IFN-γ using a targeting sgRNA (sgIL2 for IL2, sJPEGNG for IFN-γ) with or without 10 μM CNO; orange, activation using a non-targeting sgRNA with or without 10 μM CNO. The dotted line represents the detection limit of ELISA (4 pg/mL). b Activation of HBB and HBG using the hM3D-CRISPR ChaCha system measured by quantitative PCR (qPCR) with or without 10 μM CNO. c Simultaneous activation of two endogenous genes using the hM3D-CRISPR ChaCha system. Left, the plasmids of ChaCha system used for multiplexed gene regulation. Middle, a schematic overview of the ChaCha SIMO (single input, multiple output) system. Right, ELISA measure of both endogenous IL2 (orange) and IFN-γ (blue) expression and secretion using the ChaCha SIMO. For ELISA measurements, bars are the mean of three independent experiments with 7–9 technical replicates. For qPCR measurements, bars are the mean of three independent experiments that contained three technical replicates, which are then measured in technical qPCR duplicate. See Methods section for detailed experimental procedure