Scaled and efficient derivation of loss-of-function alleles in risk genes for neurodevelopmental and psychiatric disorders in human iPSCs

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. 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.

将神经发育与精神疾病（NPD，neurodevelopmental and psychiatric disorders）的遗传学发现转化为可操作的疾病生物学机制，有赖于针对NPD相关基因开展大规模且无偏倚的功能研究。本研究依托胞嘧啶碱基编辑器（CBE，cytosine base editing）系统，建立了一套在人类诱导多能干细胞（hiPSCs，human induced pluripotent stem cells）中引入提前终止密码子（iSTOP，premature stop-codons）以诱导mRNA无义介导降解（NMD，mRNA nonsense-mediated-decay）或蛋白质截短的克隆型功能丧失（LoF，loss-of-function）等位基因诱变流程。我们针对3株hiPSC细胞系中的23个NPD基因验证了该流程，在22个NPD基因中实现了重复性极佳且高效的iSTOP编辑。通过RNA测序（RNAseq），我们确认了编辑后细胞的多能性、无染色体异常以及NMD介导的转录本降解。有趣的是，针对3个精神分裂症风险基因SETD1A、TRIO与CUL1，尽管碱基编辑效率极高，但我们仅获得了杂合型LoF等位基因，这提示这些基因对细胞生长具有必需作用。我们复现了已报道的SHANK3单倍剂量不足相关神经表型，并发现CUL1-LoF会减少神经突分支与突触斑点密度。这套iSTOP流程可实现大规模、高效的NPD基因LoF诱变，产出极具价值的可共享资源。作为SSPsyGene联盟的一部分，我们的NPD的iPSC衍生神经元模型（MiNND，Model of iPSC-derived Neurons for NPD）检测与数据生成中心（ADGC，Assay and Data Generation Center）计划采用基于CBE编辑器的iSTOP方法，在多种供体遗传背景下，为150~200个NPD基因生成携带LoF等位基因的同基因hiPSC细胞系。本研究依托可显著提升hiPSCs中CBE iSTOP编辑效率的优化型报告基因编辑富集系统，建立了一套可并行处理大量基因的半自动化克隆型LoF诱变流程。我们使用3株供体hiPSC细胞系（KOLF2.2J、CW20107、MGS_CD14）对该工作流程进行了验证，在所有3株hiPSC细胞系中均获得了高效且可重复的iSTOP编辑结果。我们对工程化构建的同基因iSTOP hiPSC细胞系进行了系统性表征，包括多能性、核型、神经元分化能力，以及预期的NMD介导降解与LoF效应。