Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells

Abstract

The identification of the trans-acting factors and cis-regulatory modules that are involved in human pluripotent stem cell (hPSC) maintenance and differentiation is necessary to dissect the operating regulatory networks in these processes and thereby identify nodes where signal input will direct desired cell fate decisions in vitro or in vivo. To deconvolute these networks, we established a method to influence the differentiation state of hPSCs with a CRISPR-associated catalytically inactive dCas9 fused to an effector domain. In human embryonic stem cells, we find that the dCas9 effectors can exert positive or negative regulation on the expression of developmentally relevant genes, which can influence cell differentiation status when impinging on a key node in the regulatory network that governs the cell state. This system provides a platform for the interrogation of the underlying regulators governing specific differentiation decisions, which can then be employed to direct cellular differentiation down desired pathways.

Keywords: CRISPR; Cas9; Differentiation; Gene activation; Pluripotent stem cell; Transcriptional repression.

Fig. 1.

CRISPRa can upregulate expression of a developmentally relevant transcription factor in hESCs. (A) Genomic view of the SOX17 locus, showing the sgRNA targets, and key epigenetic marks indicating the active (H3K4me3) or repressed (H3K27me3) status, and overall accessibility (Dnase I) of the gene and its surrounding genomic area. (B) Schematic of the constitutive EF1α-regulated dCas9-VP64 and SOX17 sgRNA constructs. (C) Quantitative gene expression analysis of EF1α-regulated dCas9-VP64 cells transduced with SOX17 sgRNAs. Data are expressed as fold over hESCs ± s.d. (n=3) (D) Immunofluorescence analysis of SOX17 in EF1α-regulated dCas9-VP64 and control cells 6 days after transduction with sgRNAs. Scale bar: 100 μm.

Fig. 2.

Repression of the pluripotency network by CRISPRi-mediated downregulation of OCT4A in hESCs leads to differentiation into different cell lineages. (A) Genomic view of the OCT4 locus indicating OCT4 isoforms A and B. sgRNA targets, and key epigenetic marks indicating the active (H3K4me3) or repressed (H3K27me3) status, and overall accessibility (Dnase I) of the gene and its surrounding genomic area are indicated. (B) Schematic of the inducible TRE-regulated dCas9-KRAB and OCT4 sgRNA constructs. (C) Phase-contrast images of cells expressing unrelated (control), OCT4A or OCT4B sgRNA in TRE-regulated dCas9-KRAB cells after 6 days of doxycycline treatment. Arrow indicates morphological changes. (D,E) Immunofluorescence analysis of TRE-regulated dCas9-KRAB cells (D) or TRE-regulated dCas9 cells (F) expressing unrelated (control) or OCT4A sgRNA after 6 days of doxycycline treatment for CDX2, OCT4A, NANOG, SOX17 and T. (F) Quantitative gene expression analysis of differentiation markers CDX2, T, SOX17, SOX7 and AFP in OCT4A-158 sgRNA expressing TRE-regulated dCas9-KRAB or TRE-regulated dCas9 cells over 12 days of doxycycline treatment. Data are expressed as fold over hESCs ± s.d. (G) Quantitative gene expression analysis of differentiation markers CDX2, T, SOX17, SOX7 and AFP in OCT4A-12 sgRNA expressing TRE-regulated dCas9-KRAB or TRE-regulated dCas9 cells over 12 days of doxycycline treatment. Data are expressed as fold over hESCs ± s.d. (n=3). Scale bars: 200 μm.