Controlling transcription in human pluripotent stem cells using CRISPR-effectors

Abstract

The ability to manipulate transcription in human pluripotent stem cells (hPSCs) is fundamental for the discovery of key genes and mechanisms governing cellular state and differentiation. Recently developed CRISPR-effector systems provide a systematic approach to rapidly test gene function in mammalian cells, including hPSCs. In this review, we discuss recent advances in CRISPR-effector technologies that have been employed to control transcription through gene activation, gene repression, and epigenome engineering. We describe an application of CRISPR-effector mediated transcriptional regulation in hPSCs by targeting a synthetic promoter driving a GFP transgene, demonstrating the ease and effectiveness of CRISPR-effector mediated transcriptional regulation in hPSCs.

Keywords: CRISPR; Cas9; Human pluripotent stem cells; Transcriptional regulation; dCas9-effectors.

Figure 1. CRISPR/Cas9 effector-mediated transcriptional control

(A) Schematic depicting dCas9-effector mediated transcriptional regulation through fusion of dCas9 to transcriptional activation or repression effector domains. (B) Schematic depicting dCas9-effector mediated transcriptional regulation through recruitment of multiple antibody-fused effector domains to a dCas9-fused epitope array. (C) Schematic depicting dCas9-effector mediated transcriptional regulation through recruitment of MS2-effector fusion proteins to tethered copies of the MS2 bacteriophage coat protein-binding RNA stem loop on the 3’-end of an sgRNA. (D) Schematic depicting dCas9-effector mediated transcriptional regulation through scaffolding of a functional RNA module (such as an aptamer or lncRNA) to an sgRNA. (E) Schematic depicting dCas9-effector mediated transcriptional regulation through fusion of dCas9 to epigenetic modifiers to alter local epigenetic marks.

Figure 2. dCas9-KRAB-mediated repression of GFP in H1 CAG-GFP hPSCs

(A) Reporter construct inserted into the AAVS1 locus of H1 hPSCs via zinc finger nuclease gene editing. GFP expression is driven by a CAG (CMV-IE, chicken actin, rabbit beta globin) promoter. Target locations of CAG-specific sgRNAs (A, B, C, and D) and the transcriptional start site (TSS) are indicated. (B) Representative phase contrast and fluorescence images of H1 CAG-GFP cells demonstrating that the targeted cells maintain hPSC morphology and constitutively express GFP. Scale bar = 500 µm (C) Schematic of doxycycline-inducible dCas9-KRAB and CAG-specific sgRNA lentiviral constructs. rtTA expression is constitutively driven by the Ubiquitin C promoter (UbiC) which binds the Tet-responsive element (TRE) in the presence of doxycycline in order to activate dCas9-KRAB expression. CAG-specific sgRNA expression is constitutively driven by the U6 promoter. (D) Fluorescence and (E) flow cytometry analysis of H1 CAG-GFP cells transduced with a CAG-specific sgRNA demonstrating reduced GFP expression in an sgRNA-dependent manner following 9 days of doxycycline treatment. Scale bar = 100 µm. (F) Flow cytometry analysis of H1 CAG-GFP cells mixed with H1 cells (blue) indicating baseline fluorescence levels of both populations. Flow cytometry analysis of H1 CAG-GFP cells transduced with CAG-specific sgRNA C (green) prior to doxycycline treatment (Day 0) and acquired daily following doxycycline treatment (Day 1–Day 10). The percentage of the subset of cells that are losing GFP expression (%GFP negative) is indicated in the upper left of each time-point panel.