Fig 1.
Generation of the iKRAB ESC line.
(A) Schematic diagram shows the strategy of ICE to generate the iKRAB ESC line. FLAG-dCas9-KRAB was integrated into the downstream of the TRE element through homologous recombination. Dox-controlled rtTA drives the expression of fusion protein of FLAG-dCas9-KRAB. (B) Western blot analysis showing the inducible and reversible expression of FLAG-dCas9-KRAB protein at different time points after Dox addition or withdrawal. β-actin served as a loading control. A relative gray value quantification of dCas9-KRAB protein levels is below each lane of the band. (C, D) IF staining of Cas9 and FLAG in iKRAB ESC cultured with or without Dox. The scale bar represents 50 μm. Cas, CRISPR-associated; dCas9, deactivated Cas9; Dox, doxycycline; ESC, embryonic stem cell; ICE, inducible cassette exchange; IF, immunofluorescence; KRAB, Krüppel-associated box; rtTA, reverse tetracycline transcriptional activator; TRE, tetracyclin response element.
Fig 2.
Induced dCas9-KRAB at active gene promoters by sgRNAs or multi-gRNAs.
(A, B) RT-qPCR analysis of stable iKRAB ESCs containing sgRNA against Oct4 or multi-gRNAs against Oct4 or Nanog after 2 days of Dox induction. The binding location of each gRNA is indicated relative to the TSS of Oct4 or Nanog locus. (C) The cell morphology of Oct4 or Nanog multi-gRNAs-transduced iKRAB ESCs after 2 days of Dox induction. The scale bar represents 100 μm. The numerical values used to generate graphs in panels A and B are available in S1 Data. Cas, CRISPR-associated; dCas9, deactivated Cas9; Dox, doxycycline; ESC, embryonic stem cell; KRAB, Krüppel-associated box; RT-qPCR, reverse transcription PCR; sgRNA, single-guide RNA; TSS, transcription start site.
Fig 3.
Induced dCas9-KRAB at promoters is sufficient to maintain gene inactivation.
(A) Schematic diagram shows the dynamics of different states and marker gene expression of pluripotent stem cells cultured in different conditions. (B) RT-qPCR analysis of Fgf5 and Mll1 mRNA levels of iKRAB cells containing designated sgRNAs (with or without Dox) upon culture condition switch from 2i to FA. The binding location of each sgRNA is indicated relative to the TSS of the Fgf5 or Mll1 locus. (C) Representative IF staining of Oct4 and Nanog in iKRAB cells containing Klf5#sgRNA-2 treated with or without Dox in 2i condition or 2i switch to FA condition. The scale bar represents 100 μm. (D, G) The relative Oct4 and Nanog-positive cell numbers (normalized by DAPI+ cell numbers) are compared. (E) RT-qPCR analysis of Mll1 mRNA levels of iKRAB cells containing the same sgRNAs as (C) (with or without Dox) in FA condition. (F) Representative IF staining of Oct4 and Nanog in iKRAB cells containing Mll1#sgRNA-1 treated with or without Dox for 4 days in FA condition, followed by switch back to 2i condition. ESC constantly cultured in 2i condition was used as a control. The scale bar represents 100 μm. Data in B, D, E, and G are represented as the mean ± SD of replicates (n = 3) (*p < 0.05, **p < 0.01, ***p<0.001, ****p < 0.0001; and 2-tailed unpaired t test). The numerical values used to generate graphs in panels B, D, E, and G are available in S1 Data. ESC, embryonic stem cell; Dox, doxycycline; dCas9, deactivated Cas9; IF, immunofluorescence; RT-qPCR reverse transcription PCR; SD, standard deviation; sgRNA, single-guide RNA.
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.
Fig 5.
(A) Schematic representation of CRISPRi screens in the iKRAB ESC-derived neural cells to evaluate chromatin regulators whose depletion would resist the toxicity of sodium channel blockers A803467. (B) A map of the contribution of the top 414 sgRNAs enriched in A803467-resistant cells. PRMT2 is among the top hits. (C) Representative IF staining of Map2 in designated groups. The scale bar represents 100 μm. (D) The relative Map2-positive cell numbers are compared. (E) Validation the neuroprotective effects of PRMT inhibitor AMI-1. The scale bars represent 50 μm. (F) The relative number of viable cells in each designated group (numbers per mm2) is compared. DMSO was used as a negative control in the assay. Data in D and F 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 D and F are available in S1 Data. CRISPRi, CRISPR interference; ESC, embryonic stem cell; IF, immunofluorescence; LOF, loss-of-function; SD, standard deviation; sgRNA, single-guide RNA.
Fig 6.
Characterization of the iKRAB KI mouse and ex vivo effect.
(A) Schematic diagram shows the generation of iKRAB KI mice. (B) Genotyping PCR analysis of TRE-dCas9-KRAB and rtTA in WT and KI mice. (C) Western blot analysis of dCas9-KRAB expression in WT and iKRAB mice. β-tubulin served as a loading control. A relative gray value quantification of dCas9-KRAB protein levels is below each lane of the band. (D) Schematic diagram shows that PDLSCs from the iKRAB KI mice were introduced with sgRNA against the TSS of Runx2, followed by differentiation into osteoblasts. (E) RT-qPCR analysis showing Runx2 mRNA levels in the designated groups. Data are represented as the mean ± SD of replicates (n = 3) (****p < 0.0001; and 2-tailed unpaired t test). (F) ALP staining of cells from the designated groups. The numerical values used to generate graphs in panel E are available in S1 Data. ALP, alkaline phosphatase; KI, knock-in; PLSC, periodontal ligament stem cell; rtTA, reverse transcriptional activator; RT-qPCR, reverse transcription PCR; SD, standard deviation; sgRNA, single-guide RNA; TSS, transcription start site; WT wild type.
Fig 7.
In vivo inducible CRISPRi effect of the iKRAB KI mouse.
(A) Schematic diagram shows that AAV expressing multiplex gRNAs against Tfam locus was injected into the tibialis anterior muscle of the iKRAB KI mice, followed by 1 month of Dox induction and the subsequent analysis. (B) RT-qPCR analysis showing Tfam mRNA levels in the designated groups. (C) IF analysis of laminin expression of the tibialis anterior muscle tissue around the injection sites. GFPs mark the infected muscle fibers. The scale bar represents 50 μm. (D, E) Quantification of the cross-sectional area of the tibialis anterior muscle fibers and grip strength in designated groups (n = 6 mice). L, left; R, right. The data in B, D, and E are presented as means ± SD (n = 3 or 6) (*p < 0.05, **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, C, and E are available in S1 Data. AAV, adeno-associated viral; CRISPRi, CRISPR interference; GFP, green fluorescent protein; IF, immunofluorescence; KI, knock-in; RT-qPCR, reverse transcription PCR; SD, standard deviation.
Fig 8.
The versatility of the iKRAB KI mouse.
Primary cells isolated from the iKRAB KI mice, after being transduced by gRNAs in vitro, can be used for following ex vivo or in vivo functional studies or screens. Crossing the iKRAB KI mouse with other GEMM, we can deliver gRNAs targeting conserved regions harboring human SNPs for complex disease modeling. BMT, bone marrow transplantation; GEMM, genetically engineered mouse model; KI, knock-in; SNP, single nucleotide polymorphism.