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BRAIN Publication-derived

CRISPR/Cas9-mediated heterozygous knockout of the autism gene CHD8 and characterization of its transcriptional networks in cerebral organoids derived from iPS cells

Source Wang et al., 2017 · Albert Einstein College of Medicine · 10.1186/s13229-017-0124-1

👤 Ping Wang, Ryan Mokhtari, Erika Pedrosa, Michael Kirschenbaum, Can Bayrak, Deyou Zheng, Herbert M. Lachman ⏱ 50 days 📋 12 phases 🧫 Human iPSC

Abstract

This protocol generates cerebral organoids from CHD8 heterozygous knockout iPS cells to model autism spectrum disorder. The organoids, which resemble first-trimester telencephalon, are used to characterize transcriptional networks and identify differentially expressed genes involved in GABAergic interneuron development and neurogenesis in the context of CHD8 haploinsufficiency.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

28 steps across 12 phases

iPSC Preparation and Maintenance Day 0 to Day 1
  1. 1 Maintain iPS cell colonies on matrigel
CHD8 Knockout Generation Day 0 to Day 3 (before organoid induction)
  1. 1 Introduce CRISPR-Cas9 vector by nucleofection
Cerebral Organoid Differentiation Initiation Day 0 to Day 1
  1. 1 Pretreat iPS cells with Y27632
  2. 2 Rinse and dissociate iPS colonies
  3. 3 Collect and count dissociated cells
Aggregate Formation Day 1 to Day 2
  1. 1 Create cell aggregates using AggreWell plate
Early Organoid Differentiation (Days 1–6) Day 1 to Day 6
  1. 1 Culture aggregates in neural induction medium
Transfer to Ultra-Low Attachment Plate Day 6 to Day 18
  1. 1 Transfer aggregates to 24-well ultra-low attachment plate
  2. 2 Culture in neural induction medium without factors
  3. 3 Add N2 supplement on day 18
Plating and Maturation (Days 25–50) Day 25 to Day 50
  1. 1 Coat chamber slides with ECM proteins
  2. 2 Plate organoids on coated slides
  3. 3 Culture in Neurobasal medium with supplements
  4. 4 Detach organoids and harvest for analysis
RNA Extraction Day 50
  1. 1 Isolate total RNA from organoids
RNA Sequencing Library Preparation and Sequencing Day 50+
  1. 1 Prepare RNA-seq library
  2. 2 Perform sequencing on Illumina HiSeq 2500
RNA-seq Data Processing and Analysis Post-sequencing
  1. 1 Trim adapters and low-quality bases
  2. 2 Quantify transcript abundance using Kallisto
  3. 3 Identify differentially expressed genes using DESeq2
  4. 4 Perform Gene Ontology analysis using DAVID
  5. 5 Perform pathway analysis using IPA
Quantitative Real-Time PCR Validation Post-RNA-seq
  1. 1 Perform reverse transcription and qPCR
  2. 2 Analyze qPCR results using 2^(−ΔΔCt) method
Immunohistochemistry Day 50 or on fixed samples
  1. 1 Fix organoid samples
  2. 2 Embed samples in O.C.T compound
  3. 3 Perform immunofluorescence staining
  4. 4 Image and analyze organoid sections

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wang et al., 2017. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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