Figure 1.

A SYN-driven dCas9-KRAB-MeCP2 construct suppresses gene expression at a luciferase reporter in HEK293T cells. (A) Illustration of the dCas9-KRAB-MeCP2 suppression strategy. An sgRNA with a spacer complementary to the targeted genomic site adjacent to a PAM motif directs the dCas9-KRAB-MeCP2 transcriptional suppresser to targeted genetic loci. (B) The dual vector lentiviral construct designs. The U6 polymerase 3 promoter drives expression of the sgRNA which can be adapted to target specific genetic loci. The EF1α promoter drives expression of mCherry, useful for rapid assessment of lentiviral expression. The second construct contains the dCas9-KRAB-MeCP2 fusion driven by the neuron-selective SYN promoter. This fusion contains a FLAG epitope so that construct expression can be visualized readily by immunocytochemistry. (C) Luciferase assay confirms luciferase reporter suppression (from 4 multiplexed targeting sgRNAs) by the dCas9-KRAB-MeCP2 system in HEK293T cells relative to non-targeted (lacZ) sgRNA controls, with more robust repression than first-generation CRISPRi tools [dCas9 alone or KRAB-dCas9/EGFP; n = 8, unpaired t-test; dCas9 t₍₁₄₎ = 6.602, p = 0.000012; KRAB-dCas9/EGFP t₍₁₄₎ = 32.89, p < 0.000001; dCas9-KRAB-MeCP2 t₍₁₄₎ = 42.14, p < 0.000001]. All data are expressed as mean ± s.e.m. Individual comparisons; ****p < 0.0001.