# In vitro discovery and modeling of novel focal cortical dysplasia genes

> **NIH NIH R21** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2020 · $429,000

## Abstract

Project Summary
Most known genetic disorders arise from germline mutations that are present in every cell of the body.
However, somatic mutations that occur in neural progenitors during brain development are increasingly
recognized as a cause of focal brain malformations and epilepsy. Identifying mutations that cause these focal
malformations is extremely challenging because (1) brain tissue from patients must be available and (2) on
average only about 5% or fewer of cells in resected cortical tissue contain the causative mutation. For this
reason, targeted sequencing of genes expected to cause focal cortical dysplasia (FCD) has been the only
successful method for gene identification, and the yield is often very low. We hypothesize that an unbiased
cell culture-based, high-throughput screening approach will identify many novel FCD genes. To identify these
unknown genes, in Aim 1 we will use a CRISPR interference (CRISPRi) library screen to multiplex knockdown
of nearly every gene in cultured human induced pluripotent stem cell (iPSC)-derived, developing excitatory
cortical neurons. Positive hits will be isolated by fluorescence-activated cell sorting (FACS) for elevated
phopho-S6 (and other dysplastic cell markers) and identified by next-gen sequencing. This screen will allow us
to determine which genes, when turned off, lead to FCD-like human brain cell characteristics. In Aim 2, we will
perform multiplexed CRISPRi, single-cell RNA-sequencing (direct capture Perturb-seq) for all of the candidate
genes found in Aim 1, as well as for all known FCD genes, to both confirm the knockdown of the target gene
and compare the transcriptomic effects of each FCD gene candidate. By comparing with known FCD genes,
we will identify candidate genes with high confidence and construct an FCD transcriptomic “fingerprint” that can
be used as a resource for further understanding disease mechanisms. In Aim 3, we will apply focal CRISPRi
knockdown of DEPDC5, a known FCD gene, to cortical organoids as an in vitro human FCD model. By
combining these cutting-edge methods - high-throughput CRISPRi genetic screening, multiplexed gene
expression fingerprinting, and brain organoid cultures - these studies will advance our understanding of FCD
genetic causes and pathophysiology, providing a platform for future therapeutic studies.

## Key facts

- **NIH application ID:** 9955462
- **Project number:** 1R21NS116250-01
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Andrew M Tidball
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $429,000
- **Award type:** 1
- **Project period:** 2020-05-01 → 2022-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9955462

## Citation

> US National Institutes of Health, RePORTER application 9955462, In vitro discovery and modeling of novel focal cortical dysplasia genes (1R21NS116250-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9955462. Licensed CC0.

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