Abstract
Cerebral organoids are comprised of diverse cell types found in the developing human brain, and can be leveraged in the identification of critical cell types perturbed by genetic risk variants in common, neuropsychiatric disorders. There is great interest in developing high-throughput technologies to associate genetic variants with cell types. Here, we describe a high-throughput, quantitative approach (oFlowSeq) by utilizing CRISPR-Cas9, FACS sorting, and next-generation sequencing. Using oFlowSeq, we found that deleterious mutations in autism-associated gene KCTD13 resulted in increased proportions of Nestin+ cells and decreased proportions of TRA-1–60+ cells within mosaic cerebral organoids. We further identified that a locus-wide CRISPR-Cas9 survey of another 18 genes in the 16p11.2 locus resulted in most genes with > 2% maximum editing efficiencies for short and long indels, suggesting a high feasibility for an unbiased, locus-wide experiment using oFlowSeq. Our approach presents a novel method to identify genotype-to-cell type imbalances in an unbiased, high-throughput, quantitative manner.
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The sequencing data generated from our work will be deposited into SRA.
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Funding
This study was supported by the National Institutes of Health grants (NHGRI RM1HG008525 to GMC; NIMH R01MH113279 to GMC), and Robert Wood Johnson Foundation Grant (74178 to GMC), and startup funds by UMass Chan Medical School (ETL, YC).
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ETL, YC and GMC conceived the study. YC and ETL performed the cerebral organoid differentiation, flow cytometry and FACS experiments. PD, NJB, MS, JE, MU, CP, GMC, YC and ETL performed the analyses. LFM, MNO, MS, GY, YZ, AF, YC and ETL performed the CRISPR experiments, DNA extraction, primer design, PCR and library preparations for sequencing. MU, CP, GMC, YC and ETL supervised the research. ETL wrote the manuscript with input from all authors. All authors read and approved the final manuscript.
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Dawes, P., Murray, L.F., Olson, M.N. et al. oFlowSeq: a quantitative approach to identify protein coding mutations affecting cell type enrichment using mosaic CRISPR-Cas9 edited cerebral organoids. Hum. Genet. 142, 1281–1291 (2023). https://doi.org/10.1007/s00439-023-02534-4
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DOI: https://doi.org/10.1007/s00439-023-02534-4