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. 2021 Jun 3;2(2):100580.
doi: 10.1016/j.xpro.2021.100580. eCollection 2021 Jun 18.

Using the dCas9-KRAB system to repress gene expression in hiPSC-derived NGN2 neurons

Aiqun Li  1   2   3   4   5 , Samuel Cartwright  1   2   3 , Alex Yu  1   2   3 , Seok-Man Ho  1   3   4   5   6 , Nadine Schrode  1   3   4   5   6 , P J Michael Deans  1   3   4   5   6 , Marliette R Matos  1   3   4   5   6 , Meilin Fernandez Garcia  1   3   4   5   6 , Kayla G Townsley  1   3   4   5   6 , Bin Zhang  1   2   3 , Kristen J Brennand  1   3   4   5
Affiliations

Using the dCas9-KRAB system to repress gene expression in hiPSC-derived NGN2 neurons

Aiqun Li et al. STAR Protoc. .

Abstract

We describe a CRISPR inhibition (CRISPRi) protocol to repress endogenous gene expression (e.g., ATP6V1A) in human induced pluripotent stem cell-derived NGN2-induced glutamatergic neurons. CRISPRi enables efficient and precise gene repression of one or multiple target genes via delivering gRNA(s) to direct a dCas9-KRAB fusion protein to the gene(s) of interest. This protocol can also be adapted for gene activation and high-throughput gene manipulation, allowing assessment of the transcriptomic and phenotypic impact of candidate gene(s) associated with neurodevelopment or brain disease. For complete details on the use and execution of this protocol, please refer to Ho et al. (2017) and Wang et al. (2021).

Keywords: CRISPR; Cell Differentiation; Molecular Biology; Neuroscience; Stem Cells.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Example of CRISPR-ERA generated sgRNAs to target the promoter region for ATP6V1A knockdown (KD) E1: exon 1; E2: exon 2; TSS: transcription start site; ATG is the translation initiation codon.
Figure 2
Figure 2
Thermocycler program for annealing
Figure 3
Figure 3
An illustration of BsmBI digestion and ligation to clone an annealed oligos into lentiGuide-Hygro-mTagBFP2 For example, the 20-nucleotide (nt) sequence of ATP6V1Ai_1 gRNA (lowercase letters in gray) targeting the ATP6V1A promoter is cloned into a lentiGuide vector.
Figure 4
Figure 4
Thermocycler program for digestion and ligation
Figure 5
Figure 5
Sanger sequencing confirmation of six gRNA inserts in lentiGuide-Hygro-mTagBFP2, using the U6 primer, for ATP6V1A gene repression The flanking sequence from both sides of the insert illustrates the result of the cloning reaction.
Figure 6
Figure 6
Thermocycler program for quantitative PCR
Figure 7
Figure 7
Scheme of NGN2-neuronal differentiation from human dCas9-KRAB hiPSC-derived NPCs
Figure 8
Figure 8
Characterization of virus infection and NGN2-neuronal differentiation (A) BFP expression (Blue) of gRNA candidates 1 and 2 post virus infection. (B) Representative bright-field image of D21 NGN2 neurons. (C) Dox-induced D21 NGN2 neurons (GFP: green) express TUJ1 (red) and MAP2 (blue), pan-neuronal markers. Bars, 50 μm: A–C.
Figure 9
Figure 9
ATP6V1A RNA and protein levels reduced significantly in NGN2 neurons post gRNA perturbation (A and B) qPCR analysis (n = 4) confirms the decreased ATP6V1A RNA by gRNA candidates 1 and 2 in NGN2 neurons of two independent donors (i.e., C1 and C2). (C and D) Representative western blot and quantitative analysis (n = 4) of ATP6V1A protein levels in NGN2 neurons. β-Actin is a loading control. ANOVA with Dunnett’s test; ∗∗∗p < 0.001.
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