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1.
bioRxiv ; 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38562852

RESUMO

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.

2.
Stem Cell Reports ; 19(10): 1489-1504, 2024 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-39270650

RESUMO

Translating genetic findings for neurodevelopmental and psychiatric disorders (NPDs) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, 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 genes. Using RNA sequencing (RNA-seq), we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Despite high editing efficiency, three schizophrenia risk genes (SETD1A, TRIO, and CUL1) only had heterozygous LoF alleles, suggesting their essential roles for cell growth. We found that 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.


Assuntos
Alelos , Células-Tronco Pluripotentes Induzidas , Mutação com Perda de Função , Transtornos do Neurodesenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Transtornos do Neurodesenvolvimento/genética , Transtornos Mentais/genética , Edição de Genes , Degradação do RNAm Mediada por Códon sem Sentido , Mutagênese , Códon sem Sentido , Predisposição Genética para Doença , Sistemas CRISPR-Cas , Proteínas Culina/genética
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