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Mapping multimodal phenotypes to perturbations in cells and tissue with CRISPRmap 利用 CRISPRmap 将多模式表型映射到细胞和组织的扰动上
IF 46.9 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-07 DOI: 10.1038/s41587-024-02386-x
Jiacheng Gu, Abhishek Iyer, Ben Wesley, Angelo Taglialatela, Giuseppe Leuzzi, Sho Hangai, Aubrianna Decker, Ruoyu Gu, Naomi Klickstein, Yuanlong Shuai, Kristina Jankovic, Lucy Parker-Burns, Yinuo Jin, Jia Yi Zhang, Justin Hong, Xiang Niu, Jonathon A. Costa, Mikael G. Pezet, Jacqueline Chou, Hans-Willem Snoeck, Dan A. Landau, Elham Azizi, Edmond M. Chan, Alberto Ciccia, Jellert T. Gaublomme

Unlike sequencing-based methods, which require cell lysis, optical pooled genetic screens enable investigation of spatial phenotypes, including cell morphology, protein subcellular localization, cell–cell interactions and tissue organization, in response to targeted CRISPR perturbations. Here we report a multimodal optical pooled CRISPR screening method, which we call CRISPRmap. CRISPRmap combines in situ CRISPR guide-identifying barcode readout with multiplexed immunofluorescence and RNA detection. Barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency. CRISPRmap enables in situ barcode readout in cell types and contexts that were elusive to conventional optical pooled screening, including cultured primary cells, embryonic stem cells, induced pluripotent stem cells, derived neurons and in vivo cells in a tissue context. We conducted a screen in a breast cancer cell line of the effects of DNA damage repair gene variants on cellular responses to commonly used cancer therapies, and we show that optical phenotyping pinpoints likely pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance.

基于测序的方法需要裂解细胞,与之不同的是,光学集合基因筛选可以研究细胞形态、蛋白质亚细胞定位、细胞-细胞相互作用和组织组织等空间表型对靶向 CRISPR 干扰的响应。在这里,我们报告了一种多模式光学集合CRISPR筛选方法,我们称之为CRISPRmap。CRISPRmap 将原位 CRISPR 引导识别条形码读取与多重免疫荧光和 RNA 检测相结合。条形码通过 DNA 寡聚物的组合杂交进行检测和读出,从而提高了条形码的检测效率。CRISPRmap 能够在细胞类型和环境中进行原位条形码读取,而传统的光学集合筛选则难以实现,包括培养的原代细胞、胚胎干细胞、诱导多能干细胞、衍生神经元和组织环境中的活体细胞。我们在乳腺癌细胞系中筛选了 DNA 损伤修复基因变异对细胞对常用癌症疗法反应的影响,结果表明,光学表型能准确定位以前被归类为临床意义未知变异的可能致病的患者来源变异。
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引用次数: 0
Scalable, compressed phenotypic screening using pooled perturbations 利用集合扰动进行可扩展的压缩表型筛选
IF 46.9 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-07 DOI: 10.1038/s41587-024-02403-z
Nuo Liu, Walaa E. Kattan, Benjamin E. Mead, Conner Kummerlowe, Thomas Cheng, Sarah Ingabire, Jaime H. Cheah, Christian K. Soule, Anita Vrcic, Jane K. McIninch, Sergio Triana, Manuel Guzman, Tyler T. Dao, Joshua M. Peters, Kristen E. Lowder, Lorin Crawford, Ava P. Amini, Paul C. Blainey, William C. Hahn, Brian Cleary, Bryan Bryson, Peter S. Winter, Srivatsan Raghavan, Alex K. Shalek

High-throughput phenotypic screens using biochemical perturbations and high-content readouts are constrained by limitations of scale. To address this, we establish a method of pooling exogenous perturbations followed by computational deconvolution to reduce required sample size, labor and cost. We demonstrate the increased efficiency of compressed experimental designs compared to conventional approaches through benchmarking with a bioactive small-molecule library and a high-content imaging readout. We then apply compressed screening in two biological discovery campaigns. In the first, we use early-passage pancreatic cancer organoids to map transcriptional responses to a library of recombinant tumor microenvironment protein ligands, uncovering reproducible phenotypic shifts induced by specific ligands distinct from canonical reference signatures and correlated with clinical outcome. In the second, we identify the pleotropic modulatory effects of a chemical compound library with known mechanisms of action on primary human peripheral blood mononuclear cell immune responses. In sum, our approach empowers phenotypic screens with information-rich readouts to advance drug discovery efforts and basic biological inquiry.

使用生化扰动和高含量读数进行高通量表型筛选受到规模限制。为了解决这个问题,我们建立了一种汇集外源扰动并进行计算解卷积的方法,以减少所需的样本量、人力和成本。与传统方法相比,我们通过生物活性小分子库和高内容成像读出器进行基准测试,证明了压缩实验设计提高了效率。然后,我们在两个生物发现项目中应用了压缩筛选。在第一项研究中,我们利用早期胰腺癌器官组织绘制了重组肿瘤微环境蛋白配体库的转录反应图,发现了特定配体诱导的可重复表型转变,这种转变不同于典型的参考特征,并与临床结果相关。其次,我们确定了具有已知作用机制的化合物库对原发性人类外周血单核细胞免疫反应的多向性调节作用。总之,我们的方法赋予表型筛选以丰富的信息读数,以推进药物发现工作和基础生物学研究。
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引用次数: 0
AI meets real-world patients 人工智能与现实世界中的病人
IF 33.1 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-07 DOI: 10.1038/s41587-024-02455-1
The time has come for biotech companies to embrace machine learning for clinical trials, but they should start with compiling the data. That means grappling with real-world patient records.
生物技术公司在临床试验中采用机器学习的时机已经到来,但他们应该从汇编数据开始。这就意味着要处理真实世界的患者记录。
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引用次数: 0
Amplifying mutational profiling of extracellular vesicle mRNA with SCOPE 利用 SCOPE 放大细胞外囊泡 mRNA 的突变图谱
IF 46.9 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-07 DOI: 10.1038/s41587-024-02426-6
Jayeon Song, Mi Hyeon Cho, Hayoung Cho, Younseong Song, Sung Woon Lee, Ho Chul Nam, Tae Ho Yoon, Jong Cheol Shin, Jae-Sang Hong, Yejin Kim, Emil Ekanayake, Jueun Jeon, Dong Gil You, Sung Gap Im, Gyu-Seog Choi, Jun Seok Park, Bob C. Carter, Leonora Balaj, An Na Seo, Miles A. Miller, Soo Yeun Park, Taejoon Kang, Cesar M. Castro, Hakho Lee

Sequencing of messenger RNA (mRNA) found in extracellular vesicles (EVs) in liquid biopsies can provide clinical information such as somatic mutations, resistance profiles and tumor recurrence. Despite this, EV mRNA remains underused due to its low abundance in liquid biopsies, and large sample volumes or specialized techniques for analysis are required. Here we introduce Self-amplified and CRISPR-aided Operation to Profile EVs (SCOPE), a platform for EV mRNA detection. SCOPE leverages CRISPR-mediated recognition of target RNA using Cas13 to initiate replication and signal amplification, achieving a sub-attomolar detection limit while maintaining single-nucleotide resolution. As a proof of concept, we designed probes for key mutations in KRAS, BRAF, EGFR and IDH1 genes, optimized protocols for single-pot assays and implemented an automated device for multi-sample detection. We validated SCOPE’s ability to detect early-stage lung cancer in animal models, monitored tumor mutational burden in patients with colorectal cancer and stratified patients with glioblastoma. SCOPE can expedite readouts, augmenting the clinical use of EVs in precision oncology.

对液体活检组织细胞外囊泡 (EV) 中的信使 RNA (mRNA) 进行测序,可提供体细胞突变、耐药性特征和肿瘤复发等临床信息。尽管如此,由于EV mRNA在液体活检中的丰度较低,且需要大量样本或专业技术进行分析,因此仍未得到充分利用。在此,我们介绍一种用于 EV mRNA 检测的平台--自扩增和 CRISPR 辅助操作来剖析 EV(SCOPE)。SCOPE 利用 Cas13 介导的 CRISPR 识别目标 RNA 来启动复制和信号放大,在保持单核苷酸分辨率的同时达到亚阿托摩尔检测限。作为概念验证,我们设计了 KRAS、BRAF、表皮生长因子受体和 IDH1 基因关键突变的探针,优化了单锅检测协议,并实施了多样品检测自动装置。我们验证了 SCOPE 在动物模型中检测早期肺癌的能力,监测了结直肠癌患者的肿瘤突变负荷,并对胶质母细胞瘤患者进行了分层。SCOPE 可以加快读出速度,提高 EVs 在精准肿瘤学中的临床应用。
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引用次数: 0
Publisher Correction: Multistate and functional protein design using RoseTTAFold sequence space diffusion 出版商更正:利用 RoseTTAFold 序列空间扩散进行多态和功能蛋白质设计
IF 46.9 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-07 DOI: 10.1038/s41587-024-02456-0
Sidney Lyayuga Lisanza, Jacob Merle Gershon, Samuel W. K. Tipps, Jeremiah Nelson Sims, Lucas Arnoldt, Samuel J. Hendel, Miriam K. Simma, Ge Liu, Muna Yase, Hongwei Wu, Claire D. Tharp, Xinting Li, Alex Kang, Evans Brackenbrough, Asim K. Bera, Stacey Gerben, Bruce J. Wittmann, Andrew C. McShan, David Baker

Correction to: Nature Biotechnology https://doi.org/10.1038/s41587-024-02395-w, published online 25 September 2024.

更正为自然生物技术 https://doi.org/10.1038/s41587-024-02395-w,2024 年 9 月 25 日在线发表。
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引用次数: 0
A CRISPR-based method for detecting mRNA in extracellular vesicles 基于 CRISPR 的细胞外囊泡 mRNA 检测方法
IF 46.9 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-07 DOI: 10.1038/s41587-024-02440-8
A CRISPR-based assay both recognizes and amplifies target mRNA, achieving sub-attomolar sensitivity with single-nucleotide resolution. This method enables the detection of low-abundance mRNA in extracellular vesicles, providing clinically relevant information for precision oncology.
一种基于 CRISPR 的检测方法既能识别又能扩增目标 mRNA,实现了亚阿托摩尔灵敏度和单核苷酸分辨率。这种方法能够检测细胞外囊泡中的低丰度 mRNA,为精准肿瘤学提供临床相关信息。
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引用次数: 0
Advances in optical pooled screening to map spatial complexity 用于绘制空间复杂性图谱的光学集合筛选技术取得进展
IF 46.9 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-07 DOI: 10.1038/s41587-024-02434-6
Maurice Kahnwald, Marius Mählen, Koen C. Oost, Prisca Liberali
Two major advances in optical pooled screening improve substantially on sensitivity and robustness, expanding its applicability to a broader range of biological contexts.
光学集合筛选的两大进展大大提高了灵敏度和稳健性,使其适用于更广泛的生物环境。
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引用次数: 0
Multiplexed, image-based pooled screens in primary cells and tissues with PerturbView 利用 PerturbView 对原代细胞和组织进行基于图像的多重集合筛选
IF 46.9 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-07 DOI: 10.1038/s41587-024-02391-0
Takamasa Kudo, Ana M. Meireles, Reuben Moncada, Yushu Chen, Ping Wu, Joshua Gould, Xiaoyu Hu, Opher Kornfeld, Rajiv Jesudason, Conrad Foo, Burkhard Höckendorf, Hector Corrada Bravo, Jason P. Town, Runmin Wei, Antonio Rios, Vineethkrishna Chandrasekar, Melanie Heinlein, Amy S. Chuong, Shuangyi Cai, Cherry Sakura Lu, Paula Coelho, Monika Mis, Cemre Celen, Noelyn Kljavin, Jian Jiang, David Richmond, Pratiksha Thakore, Elia Benito-Gutiérrez, Kathryn Geiger-Schuller, Jose Sergio Hleap, Nobuhiko Kayagaki, Felipe de Sousa e Melo, Lisa McGinnis, Bo Li, Avtar Singh, Levi Garraway, Orit Rozenblatt-Rosen, Aviv Regev, Eric Lubeck

Optical pooled screening (OPS) is a scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture due to limitations associated with in situ sequencing of perturbation barcodes. Here, we develop PerturbView, an OPS technology that leverages in vitro transcription to amplify barcodes before in situ sequencing, enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues. We demonstrate PerturbView in induced pluripotent stem cell-derived neurons, primary immune cells and tumor tissue sections from animal models. In a screen of immune signaling pathways in primary bone marrow-derived macrophages, PerturbView uncovered both known and novel regulators of NF-κB signaling. Furthermore, we combine PerturbView with spatial transcriptomics in tissue sections from a mouse xenograft model, paving the way to in situ screens with rich optical and transcriptomic phenotypes. PerturbView broadens the scope of OPS to a wide range of models and applications.

光学集合筛选(OPS)是一种将基于图像的表型与细胞扰动联系起来的可扩展方法。然而,由于扰动条形码原位测序的局限性,迄今为止,这种方法仅限于在培养中的癌细胞系中进行相对低倍数的表型读出。在这里,我们开发了 PerturbView,这是一种 OPS 技术,它利用体外转录技术在原位测序前扩增条形码,从而能在包括原代细胞和组织在内的不同系统中筛选出高度复用的表型读数。我们在诱导多能干细胞衍生的神经元、原代免疫细胞和动物模型的肿瘤组织切片中展示了 PerturbView。在对原代骨髓巨噬细胞的免疫信号通路进行筛选时,PerturbView 发现了 NF-κB 信号传导的已知和新型调节因子。此外,我们将 PerturbView 与小鼠异种移植模型组织切片中的空间转录组学相结合,为具有丰富光学和转录组表型的原位筛选铺平了道路。PerturbView 拓宽了 OPS 的范围,使其适用于各种模型和应用。
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引用次数: 0
Bacteria displaying cytokines heat up the tumor microenvironment 显示细胞因子的细菌使肿瘤微环境升温
IF 46.9 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-04 DOI: 10.1038/s41587-024-02429-3
Marine Fidelle, Laurence Zitvogel
Escherichia coli engineered to display cytokines destroy hard-to-treat tumors by boosting the activity of local native and adoptive immune effector cells.
大肠杆菌经改造后可显示细胞因子,通过增强本地和收养性免疫效应细胞的活性来摧毁难以治疗的肿瘤。
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引用次数: 0
Non-pathogenic E. coli displaying decoy-resistant IL18 mutein boosts anti-tumor and CAR NK cell responses 显示抗诱饵 IL18 静音蛋白的非致病性大肠杆菌可增强抗肿瘤和 CAR NK 细胞反应
IF 46.9 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-04 DOI: 10.1038/s41587-024-02418-6
Shaobo Yang, Michal Sheffer, Isabel E. Kaplan, Zongqi Wang, Mubin Tarannum, Khanhlinh Dinh, Yasmin Abdulhamid, Eden Bobilev, Roman Shapiro, Rebecca Porter, Robert Soiffer, Jerome Ritz, John Koreth, Yun Wei, Peiru Chen, Ke Zhang, Valeria Márquez-Pellegrin, Shanna Bonanno, Neel Joshi, Ming Guan, Mengdi Yang, Deng Li, Chiara Bellini, Fuguo Liu, Jianzhu Chen, Catherine J. Wu, David Barbie, Jiahe Li, Rizwan Romee

The tumor microenvironment can inhibit the efficacy of cancer therapies through mechanisms such as poor trafficking and exhaustion of immune cells. Here, to address this challenge, we exploited the safety, tumor tropism and ease of genetic manipulation of non-pathogenic Escherichia coli (E. coli) to deliver key immune-activating cytokines to tumors via surface display on the outer membrane of E. coli K-12 DH5α. Non-pathogenic E. coli expressing murine decoy-resistant IL18 mutein (DR18) induced robust CD8+ T and natural killer (NK) cell-dependent immune responses and suppressed tumor progression in immune-competent colorectal carcinoma and melanoma mouse models. E. coli K-12 DH5α engineered to display human DR18 potently activated mesothelin-targeting chimeric antigen receptor (CAR) NK cells and enhance their trafficking into tumors, which extended survival in an NK cell treatment-resistant mesothelioma xenograft model by enhancing TNF signaling and upregulating NK activation markers. Our live bacteria-based immunotherapeutic system safely and effectively induces potent anti-tumor responses in treatment-resistant solid tumors, motivating further evaluation of this approach in the clinic.

肿瘤微环境可通过免疫细胞的贩运不畅和耗竭等机制抑制癌症疗法的疗效。在这里,为了应对这一挑战,我们利用非致病性大肠杆菌(E. coli)的安全性、肿瘤滋养性和易于遗传操作的特点,通过在大肠杆菌 K-12 DH5α 外膜上的表面显示,向肿瘤输送关键的免疫激活细胞因子。表达小鼠抗诱饵 IL18 静音素(DR18)的非致病性大肠杆菌诱导了强大的 CD8+ T 和自然杀伤(NK)细胞依赖性免疫反应,并抑制了免疫功能正常的结直肠癌和黑色素瘤小鼠模型的肿瘤进展。大肠杆菌 K-12 DH5α 经改造后可显示人类 DR18,它能有效激活间皮素靶向嵌合抗原受体(CAR)NK 细胞,并增强其向肿瘤的迁移,通过增强 TNF 信号传导和上调 NK 激活标记物,延长了 NK 细胞治疗耐药间皮瘤异种移植模型的存活时间。我们基于活细菌的免疫治疗系统能安全有效地诱导耐药实体瘤产生强效抗肿瘤反应,促使我们在临床中进一步评估这种方法。
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引用次数: 0
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Nature biotechnology
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