Developing high-throughput genetic perturbation strategies for single cells in cancer organoids

项目总结 要解决化疗耐药和经常复发的异质癌症的复杂性 或者转移，我们建议开发一套基于多学科创新的工具，结合综合 生物学、癌症有机体技术和生物信息学。这些合成工具可以为Reporter添加注释 用于癌症异质性和复发发展的有机化合物(StarOrchard)包括：合成启动子 万花筒有机化合物的激活重组(SPEKO)，组合遗传学En MASSE(CombiGEM)和单细胞RNA测序全景图(Scanorama)。斯帕科可以注解 通过荧光蛋白表达文库在活细胞中表达异质性肿瘤群体 彩色肿瘤器官。CombiGEM可以通过大规模、 大规模平行、无偏见的组合遗传筛选。Scanorama可以整合分析 通过复杂的生物信息学算法对单细胞转录组的大数据集进行分析。这些工具 重点关注条形码策略，以实现对含有 独特的遗传异常，并极大地扩展了下一代癌症模型的应用 (NGCMS)用于癌症机制研究或治疗发现。StarOrchard工具支持 在不破坏细胞的情况下，注解的异质性肿瘤表型中的定向遗传扰动 测序。这些工具将应用于大量和多样化的NGCM以优化实验 协议。为了确保成功，我们召集了一支杰出的团队：Pi Timothy K.Lu，医学博士，拥有 对合成生物学工具做出了惊人的原创性贡献，使高通量基因 癌症细胞药物依赖的审问；皮奥默·耶尔马兹，医学博士，在癌症方面拥有丰富的专业知识 并开发了新的技术来维持患者来源的结肠癌 用于活体建模的有机化合物；以及Pi Bonnie Berger博士将利用她在生物信息学和 她的Scanorama算法基于动态单细胞RNA集成所有肿瘤类型的数据 测序(ScRNAseq)。我们还得到了癌症生物学和各种癌症方面的领先专家的支持 癌症研究的基础科学和临床肿瘤学前沿的类型。集体 整个团队的承诺和多学科贡献确保了一个公开的 分布式研究工具集，加速癌症生物学和治疗发现的进步

PROJECT SUMMARY To address the complexity of heterogeneous cancers that are resistant to chemotherapy and frequently recur or metastasize, we propose to develop a set of tools based on multidisciplinary innovations combining Synthetic Biology, Cancer Organoid Technology, and Bioinformatics. These Synthetic Tools to Annotate Reporter Organoids for Cancer Heterogeneity and Recurrence Development (StarOrchard) include: Synthetic Promoter Activated Recombination of Kaleidoscopic Organoids (SPARKO), Combinatorial Genetics En Masse (CombiGEM), and single-cell RNA sequencing panorama (Scanorama). SPARKO can annotate heterogeneous cancer populations in living cells via fluorescent protein expression libraries to make multi- colored tumor organoids. CombiGEM can rapidly identify potential therapeutic targets via large-scale, massively parallel, and unbiased combinatorial genetic screens. Scanorama can integrate the analysis of large datasets of single-cell transcriptomics via sophisticated bioinformatics algorithms. These tools focus on barcoding strategies to enable accurate tracking and analysis of individual tumor cells that harbor distinct genetic aberrations, and substantially expand the utility of the Next Generation Cancer Models (NGCMs) for cancer mechanistic investigations or therapeutic discovery. The StarOrchard tools enable targeted genetic perturbations in annotated heterogeneous tumor phenotypes without destroying cells for sequencing. These tools will be applied to a large number and variety of NGCMs to optimize experimental protocol. To ensure success, we have convened an outstanding team: PI Timothy K. Lu, MD, PhD, has made strikingly original contributions to Synthetic Biology tools that enable high-throughput genetic interrogation of cancer cell drug dependency; PI Ömer Yilmaz, MD, PhD, has extensive expertise in cancers of the gastrointestinal tract and has developed novel technologies to maintain patient-derived colon cancer organoids for in vivo modeling; and PI Bonnie Berger, PhD, will use her expertise in bioinformatics and her Scanorama algorithm to integrate data across all tumor types based on dynamic single cell RNA sequencing (scRNAseq). We are also supported by leading experts in cancer biology and various cancer types at both the basic science and clinical oncology frontiers of cancer research. The collective commitment and multidisciplinary contributions of the entire team ensure the establishment of an openly distributed investigative tool set that accelerates advancements in cancer biology and therapeutic discovery