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| Status |
Public on Sep 17, 2024 |
| Title |
Scaled and efficient derivation of loss-of-function alleles in risk genes for neurodevelopmental and psychiatric disorders in human iPSCs |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Translating genetic findings for neurodevelopmental and psychiatric disorders (NPD) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, here we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop-codons (iSTOP) that lead to mRNA nonsense-mediated-decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 NPD genes. Using RNAseq, we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Interestingly, for three schizophrenia risk genes (SETD1A, TRIO, CUL1), despite the high efficiency of base editing, we only obtained heterozygous LoF alleles, suggesting their essential roles for cell growth. We replicated the reported neural phenotypes of SHANK3-haploinsufficiency and found CUL1-LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.
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| Overall design |
As part of the SSPsyGene Consortium, our Assay and Data Generation Center (ADGC) for the Model of iPSC-derived Neurons for NPD (MiNND) aims to employ the CBE editor-based iSTOP approach to generate isogenic hiPSC lines carrying LoF alleles for about 150-200 NPD genes on multiple donor genetic backgrounds. Here, leveraging an improved reporter gene editing enrichment system that can substantially increase the CBE iSTOP editing efficiency in hiPSC, we established a semi-automated pipeline for parallel and efficient clonal LoF mutagenesis of a large number of genes. We tested the workflow on 23 NPD genes with 3 donor hiPSC lines (KOLF2.2J, CW20107, MGS_CD14). We obtained high and reproducible iSTOP editing efficiency across all three hiPSC lines. We systematically characterized the engineered isogenic iSTOP hiPSC lines for pluripotency, karyotyping, neuron differentiation capacity, and the expected NMD and LoF.
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| Web link |
https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(24)00241-8
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| Contributor(s) |
Duan J, Zhang S, Pang ZP |
| Citation missing |
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| Submission date |
Mar 26, 2024 |
| Last update date |
Sep 17, 2024 |
| Contact name |
Jubao Duan |
| E-mail(s) |
jduan69@gmail.com
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| Phone |
(224) 364-7564
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| Organization name |
NorthShore University HealthSystem
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| Department |
Center for Psychiatric Genetics
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| Lab |
Unit of Functional Genomics in Psychiatry
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| Street address |
1001 University Place
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| City |
Evanston |
| State/province |
IL |
| ZIP/Postal code |
60201 |
| Country |
USA |
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| Platforms (1) |
| GPL34281 |
Illumina NovaSeq X (Homo sapiens) |
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| Samples (69)
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| Relations |
| BioProject |
PRJNA1091873 |
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