Evaluating Synthetic Activation and Repression of Neuropsychiatric-Related Genes in hiPSC-Derived NPCs, Neurons, and Astrocytes

Abstract

Modulation of transcription, either synthetic activation or repression, via dCas9-fusion proteins is a relatively new methodology with the potential to facilitate high-throughput up- or downregulation studies of gene function. Genetic studies of neurodevelopmental disorders have identified a growing list of risk variants, including both common single-nucleotide variants and rare copy-number variations, many of which are associated with genes having limited functional annotations. By applying a CRISPR-mediated gene-activation/repression platform to populations of human-induced pluripotent stem cell-derived neural progenitor cells, neurons, and astrocytes, we demonstrate that it is possible to manipulate endogenous expression levels of candidate neuropsychiatric risk genes across these three cell types. Although proof-of-concept studies using catalytically inactive Cas9-fusion proteins to modulate transcription have been reported, here we present a detailed survey of the reproducibility of gRNA positional effects across a variety of neurodevelopmental disorder-relevant risk genes, donors, neural cell types, and dCas9 effectors.

Keywords: CRISPR; dCas9-KRAB; dCas9-VP64; dCas9-VPR; human-induced pluripotent stem cell; neural progenitor cell; transcriptional modulation.

Figure 1

Experimental Platform to Evaluate dCas9-Mediated Manipulation of Gene Expression in hiPSC-Derived NPCs, Neurons, and Astrocytes (A) Experimental schematic for the generation of hiPSC-derived NPCs, neurons, and astrocytes, including the administration of lentiviral dCas9 and gRNA vectors. (B) Experimental time line for lentiviral transduction (and antibiotic selection) of hiPSC-derived NPCs, neurons, and astrocytes with lentiviral dCas9 and gRNA vectors. (C) gRNA locations (green, blue, and yellow) relative to the TSS of KCTD13, TAOK2, NRXN1, SNAP91, and CLCN3.

Figure 2

Activation of Neuropsychiatric Disorder Risk Genes in hiPSC-Derived NPCs and NGN2 Neurons from Three Individuals (A–F) Normalized relative mRNA levels (compared with no gRNA or scrambled gRNA control as indicated [gray]) following transduction of dCas9^VP64 (A, B, C, and E) and dCas9^VPR (C, D, and F) in NPCs (A, B, C, and E) and 8-day-old NGN2 neurons (D and F) with lentivirus-expressing gRNAs targeted to three different locations (green, blue, and yellow) upstream of the TSS for KCTD13 (A), TAOK2 (B), NRXN1 (C), SNAP91 (D and E), and CLCN3 (F). C1–C3 indicates hiPSC lines from three independent male controls (see Table S1 for more information); each biological replicate is depicted by a circle. Data are presented as means ± SEM (bar graph) from at least three independent biological replicates. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

Figure 3

Repression of Neuropsychiatric Disorder Risk Genes in hiPSC-Derived NPCs and NGN2 Neurons from Three Individuals (A–F) Normalized relative mRNA levels (compared with no gRNA control [gray]) following transduction of dCas9^KRAB NPCs (A, B, and E), 8-day-old (C and F), and 20-day-old (D) NGN2 neurons with lentivirus-expressing gRNAs targeted to different locations (green, blue, and yellow) upstream of the TSS for KCTD13 (A), TAOK2 (B), SNAP91 (C, D, and E), and CLCN3 (F). C1–C3 indicates hiPSC lines from three independent male controls (see Table S1 for more information); each biological replicate is depicted by a circle. Data are presented as means ± SEM (bar graph) from at least three independent biological replicates. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

Figure 4

Activation of Neuropsychiatric Disorder Risk Genes in NPC-Derived Astrocytes from Three Individuals (A) Representative FACS validation of NPC-astrocytes, using antibodies for GFAP (left), S100β (middle), and EAAT1 (right). (B) Representative immunofluorescent image of NPC-astrocytes stained with vimentin (green) and DAPI (blue). Scale bar, 20 μm. (C) FACS validation of Cas9 protein levels in dCas9^-VP64 NPC-astrocytes. (D and E) Normalized relative mRNA levels (compared with no gRNA control [gray]) following transduction of dCas9^VP64 NPC-astrocytes with lentivirus-expressing gRNAs targeted to three different locations (green, blue, and yellow) upstream of the TSS for KCTD13 (D) and SNAP91 (E). C1–C3 indicates hiPSC lines from three independent male controls (see Table S1 for more information); each biological replicate is depicted by a circle. Data are presented as means ± SEM (bar graph) from at least three independent biological replicates. ^∗p < 0.05, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

Figure 5

Single and Multiplexed IVT gRNA-Mediated Transcriptional Modulation (A) Transient expression of IVT gRNAs for transcriptional modulation of SNAP91 (1 gRNA), CEP162 (six pooled gRNAs), and FUT9 (ten pooled gRNAs). Normalized relative mRNA levels (compared with GFP [SNAP91] or ten gRNAs targeting a nearby non-promoter non-coding region [CEP162 and FUT9]) following transfection of dCas9^VP64 NPCs with IVT gRNAs targeted upstream of the TSS for SNAP91, CEP162, and FUT9. (B) Multiplexed pools of ten, five (two independent pools), and one IVT gRNA(s) for transcriptional modulation of FUT9. Data are presented as means ± SEM (bar graph) from at least three independent biological replicates. Each biological replicate is depicted by a circle. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.