An Improved CRISPR/dCas9 Interference Tool for Neuronal Gene Suppression

Abstract

The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Previously, we optimized a dual lentivirus expression system to express CRISPR-based activation machinery in post-mitotic neurons. Here we used a similar strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. We find that lentiviral delivery of a dCas9-KRAB-MeCP2 construct driven by the neuron-selective human synapsin promoter enabled transgene expression in primary rat neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Brain-derived neurotrophic factor (Bdnf) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system.

Keywords: CRISPRi; KRAB-MeCP2; dCas9; gene regulation; neurons; transcription.

Figure 1

A SYN-driven dCas9-KRAB-MeCP2 construct suppresses gene expression at a luciferase reporter in HEK293T cells. (A) Illustration of the dCas9-KRAB-MeCP2 suppression strategy. An sgRNA with a spacer complementary to the targeted genomic site adjacent to a PAM motif directs the dCas9-KRAB-MeCP2 transcriptional suppresser to targeted genetic loci. (B) The dual vector lentiviral construct designs. The U6 polymerase 3 promoter drives expression of the sgRNA which can be adapted to target specific genetic loci. The EF1α promoter drives expression of mCherry, useful for rapid assessment of lentiviral expression. The second construct contains the dCas9-KRAB-MeCP2 fusion driven by the neuron-selective SYN promoter. This fusion contains a FLAG epitope so that construct expression can be visualized readily by immunocytochemistry. (C) Luciferase assay confirms luciferase reporter suppression (from 4 multiplexed targeting sgRNAs) by the dCas9-KRAB-MeCP2 system in HEK293T cells relative to non-targeted (lacZ) sgRNA controls, with more robust repression than first-generation CRISPRi tools [dCas9 alone or KRAB-dCas9/EGFP; n = 8, unpaired t-test; dCas9 t₍₁₄₎ = 6.602, p = 0.000012; KRAB-dCas9/EGFP t₍₁₄₎ = 32.89, p < 0.000001; dCas9-KRAB-MeCP2 t₍₁₄₎ = 42.14, p < 0.000001]. All data are expressed as mean ± s.e.m. Individual comparisons; ****p < 0.0001.

Figure 2

dCas9-KRAB-MeCP2 is capable of strong gene suppression at multiple genes in primary neuronal cultures. (A) Immunocytochemistry demonstrating expression of the lentiviral SYN-driven dCas9-KRAB-MeCP2 construct in primary rat striatal cultures. Scale bar = 200 μm. (B) dCas9-KRAB-MeCP2 induces targeted gene suppression at Fosb (recruited by an individual sgRNA) relative to lacZ sgRNA controls in primary rat hippocampal cultures as revealed by RT-qPCR. There was no main effect of viral genome copies per cell [n = 2, Two-Way ANOVA; sgRNA F_(1,6) = 19.17, p = 0.0047; viral genomic copies per cell F_(2,6) = 0.2380, p = 0.7953, interaction F_(2,6) = 0.2380, p = 0.7953]. (C) RT-qPCR demonstrates targeted gene suppression in striatal cultures relative to non-targeted controls across multiple genes, with either 6 sgRNAs targeting Kmt2b, 1 sgRNA targeting Reln, or 3 sgRNAs targeting Npy [n = 3–6, Two-Way ANOVA; left (Kmt2b expression): lacZ vs targeted sgRNA(s) F_(1,18) = 8.088, p = 0.0108, and gene targeted F_(2,18) = 5.400, p = 0.0146, Sidak's multiple comparisons test: Kmt2b t₍₁₈₎ = 3.642, p = 0.0056; Reln t₍₁₈₎ = 0.5278, p = 0.9380; Npy t₍₁₈₎ = 1.228, p = 0.5529; middle (Reln expression); lacZ vs. targeted sgRNA(s) F_(1,18) = 61.82, p < 0.0001, and gene targeted F_(2,18) = 15.92, p = 0.0001, Sidak's multiple comparisons test: Kmt2b t₍₁₈₎ = 2.511, p = 0.0640; Reln t₍₁₈₎ = 11.17, p < 0.0001; Npy t₍₁₈₎ = 2.022, p = 0.1648; right (Npy expression); lacZ vs targeted sgRNA(s) F_(1,18) = 8.160, p = 0.0105, and gene targeted F_(2,18) = 12.67, p = 0.0004, Sidak's multiple comparisons test: Kmt2b t₍₁₈₎ = 1.237, p = 0.5470; Reln t₍₁₈₎ = 0.4606, p = 0.9573; Npy t₍₁₈₎ = 5.428, p = 0.0001]. All data are expressed as mean ± s.e.m. Individual comparisons, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

dCas9-KRAB-MeCP2 induces targeted transcript specific Bdnf gene repression in primary rat hippocampal cultures. (A) Complex structure of the rat Bdnf gene produces spliced transcripts from multiple promoter regions using non-coding exons (I-IXa) and a common coding exon (IX). shRNAs (left) or sgRNAs (right) can be designed for targeted transcript suppression. (B) shRNA targeting the Bdnf I variant did not significantly decrease its expression as assessed by RT-qPCR, while shRNA designed against the Bdnf IV variant increased expression of the Bdnf I variant [left, Bdnf I RT-qPCR: n = 4–6, One-Way ANOVA, F_(2,11) = 32.55, p < 0.0001. Middle, Bdnf IV RT-qPCR: n = 4–6, One-Way ANOVA, F_(2,11) = 25.19, p < 0.0001]. Targeting either variant did not alter the expression levels of total Bdnf as measured using qPCR primers designed for the Bdnf IX common coding region [right, Bdnf IX RT-qPCR: n = 4–6, One-Way ANOVA, F_(2,11) = 7.266, p = 0.0097]. (C) Individual sgRNAs designed upstream of the Bdnf I and Bdnf IV exons recruit dCas9-KRAB-MeCP2 to induce transcript specific gene repression of the Bdnf I and Bdnf IV transcript variants as assessed by RT-qPCR. Both sgRNAs resulted in a significant decrease in total Bdnf IX expression [left, Bdnf I RT-qPCR: n = 6, One-Way ANOVA, F_(2,15) = 41.87, p < 0.0001. Middle, Bdnf IV RT-qPCR: n = 6, One-Way ANOVA, F_(2,15) = 181.9, p < 0.0001. Right, Bdnf IX RT-qPCR: n = 6, one-way ANOVA, F_(2,15) = 74.56, p < 0.0001]. Tukey's multiple comparisons test was used for individual comparisons. All data are expressed as mean ± s.e.m. Individual comparisons; ***p < 0.001, ****p < 0.0001.