Large-Scale CRISPR Screening in Primary Human 3D Gastric Organoids Enables Comprehensive Dissection of Gene-Drug Interactions [ATAC-seq]

Investigating gene-drug interactions is critical for advancing personalized medicine by revealing how genetic variations shape individual responses to medications. To achieve this goal, it is essential to develop a robust human functional genomics system that can accurately recapitulate normal physiology and disease while effectively accounting for the complexity and heterogeneity among individuals. We explored this potential in primary 3D human gastric organoids by implementing a wide range of large-scale CRISPR-based genetic screens, including CRISPR knockout, CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), and single-cell CRISPR screens. Our cisplatin sensitization screens targeting 1,952 DNA-binding proteins identified numerous known genes associated with DNA repair pathways, along with previously unrecognized cisplatin sensitization loci. Individual validation of top hits further confirmed the robustness of the screening results. Furthermore, using multiplexed single-cell CRISPR screens to simultaneously extract individual sgRNAs and single-cell transcriptomes, we uncovered a convergence within DNA repair pathways in cisplatin-treated organoids compared to cisplatin-naïve organoids. These methods distinguished high-dimensional synergistic effects between sgRNAs and drug perturbations by leveraging each cell's gene expression profiles, improving the resolution to link genotypes with drug response phenotypes in organoids. Finally, we identified TAF6L as a novel cisplatin sensitization gene. Mechanistically, TAF6L is crucial for the proliferation of cell recovery following cisplatin-induced DNA damage response. In summary, diverse CRISPR-based functional genomics platforms can be successfully applied to human organoids, revealing new gene-drug interactions with implications for human disease. In order to investigate TAF6L-associated chromatin accessibility, we established TAF6L knockdown, TAG6L-GFP overexpression and their control human gastric organoid lines followed by ATAC-seq analyses.

探究基因-药物相互作用，对于推动精准医学发展至关重要，此类研究可揭示遗传变异如何塑造个体对药物的应答反应。为达成这一目标，亟需构建一套稳健的人类功能基因组学（functional genomics）系统，该系统能够精准复刻正常生理状态与疾病状态，同时有效兼顾个体间的复杂性与异质性。我们在原代三维人类胃类器官（3D human gastric organoids）中探索了这一应用潜力，通过实施一系列大规模基于CRISPR的遗传筛选，包括CRISPR敲除（CRISPR knockout）、CRISPR干扰（CRISPR interference, CRISPRi）、CRISPR激活（CRISPR activation, CRISPRa）以及单细胞CRISPR筛选。我们针对1952个DNA结合蛋白开展了顺铂致敏筛选，从中鉴定出众多与DNA修复通路相关的已知基因，同时发现了此前未被报道的顺铂致敏位点。对筛选得到的顶级候选命中基因进行个体验证，进一步证实了筛选结果的可靠性。此外，我们利用多重单细胞CRISPR筛选技术，同步获取单个向导RNA（single-guide RNA, sgRNA）的信息与单细胞转录组数据，对比顺铂处理与未接受顺铂处理的类器官，发现顺铂处理后的类器官中存在DNA修复通路的趋同调控现象。该方法借助每个细胞的基因表达谱，解析了向导RNA与药物扰动之间的高维协同效应，提升了在类器官模型中关联基因型与药物应答表型的分辨率。最终，我们鉴定出TAF6L作为一种新型顺铂致敏基因。从机制层面来看，TAF6L对于顺铂诱导的DNA损伤应答（DNA damage response）后细胞的增殖恢复至关重要。综上，各类基于CRISPR的功能基因组学平台可成功应用于人类类器官研究，进而揭示出与人类疾病相关的新型基因-药物相互作用。为探究TAF6L相关的染色质可及性，我们构建了TAF6L敲低、TAG6L-GFP过表达及其对应的对照人类胃类器官系，并随后开展了ATAC-seq（assay for transposase-accessible chromatin using sequencing）分析。