Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model
Fig 4
Induced dCas9-KRAB at enhancers is sufficient to maintain gene inactivation in response to activation signals.
(A) Schematic diagram shows the PE of Oct4 is activated upon culture condition switch from 2i to FA. Dox-induced dCas9-KRAB is tethered to the PE of Oct4 in 2i condition, and Oct4 expression is to be tested in SL and FA conditions. (B) RT-qPCR analysis of Oct4 mRNA levels of iKRAB cells containing designated sgRNAs (with or without Dox) upon culture condition switch from 2i to SL or FA. The binding location of each sgRNA is indicated relative to the PE of Oct4 locus. (C) Representative IF staining of Oct4 and Nanog in designated conditions. The scale bar represents 20 μm. (D, I) The relative Oct4, Nanog, or Map2-positive cell numbers are compared. (E) Schematic diagram shows iKRAB cells containing designated sgRNAs the PE of Sox2 are differentiated to NPC and neuron. Dox was added before NPC stage together with RA. (F) RT-qPCR analysis of Sox2 mRNA levels in NPC (8 days) from the group with or without treatment. (G) RT-qPCR analysis of 2 neuron marker genes in neuron (12 days) from the group with or without treatment. (H) Representative IF staining of Map2 in neuron (12 days) from the group with or without treatment. The scale bar represents 50 μm. (J) A model for the temporal control of enhancer or promoter by the timing of Dox addition during ESC differentiation. Data in B, D, F, G, and I are represented as the mean ± SD of replicates (n = 3 or 4) (**p < 0.01, ***p < 0.001, ****p < 0.0001; and 2-tailed unpaired t test). The numerical values used to generate graphs in panel B, D, F, G, and I are available in S1 Data. Dox, doxycycline; dCas9, deactivated Cas9; IF, immunofluorescence; KRAB, Krüppel-associated box; NPC, neural progenitor cell; PE, proximal enhancer; RT-qPCR, reverse transcription PCR; sgRNA, single-guide RNA.