Colorectal cancer (CRC) is characterized by prominent genetic and phenotypic heterogeneity between patients. To facilitate high-throughput genetic testing and functional identification of tumor drivers, we developed a platform for pooled CRISPR-Cas9 screening in human colon organoids. Using transforming growth factor β (TGF-β) resistance as a paradigm to establish sensitivity and scalability in vitro, we identified optimal conditions and strict guide RNA (gRNA) requirements for screening in 3D organoids. We then screened a pan-cancer tumor suppressor gene (TSG) library in pre-malignant organoids with APC;KRAS mutations, which were xenografted to study clonal advantages in context of a complex tumor microenvironment. We identified TGFBR2 as the most prevalent TSG, followed by known and previously uncharacterized mediators of CRC growth. gRNAs were validated in a secondary screen using unique molecular identifiers (UMIs) to adjust for clonal drift and to distinguish clone size and abundance. Together, these findings highlight a powerful organoid-based platform for pooled CRISPR-Cas9 screening for patient-specific functional genomics.
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http://dx.doi.org/10.1016/j.stem.2020.04.003 | DOI Listing |
Plants (Basel)
December 2024
State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of the National Forestry and Grassland Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China.
This study aimed to establish a CRISPR/Cas9 gene-editing system for (Lamb.) Carr. (Japanese larch).
View Article and Find Full Text PDFEMBO Rep
January 2025
Cellular and Molecular Physiology, Institute of Systems Molecular and Integrative Biology, University of Liverpool, Crown St, Liverpool, L69 3BX, UK.
Cancer cells often display centrosome amplification, requiring the kinesin KIFC1/HSET for centrosome clustering to prevent multipolar spindles and cell death. In parallel siRNA screens of deubiquitinase enzymes, we identify OTUD6B as a positive regulator of KIFC1 expression that is required for centrosome clustering in triple-negative breast cancer (TNBC) cells. OTUD6B can localise to centrosomes and the mitotic spindle and interacts with KIFC1.
View Article and Find Full Text PDFZhong Nan Da Xue Xue Bao Yi Xue Ban
August 2024
Department of Parasitology, School of Basic Medical Sciences, Central South University, Changsha 410013.
Objectives: Toxoplasmosis is a zoonotic parasitic disease caused by (), which can lead to complications such as encephalitis and ocular toxoplasmosis. The disease becomes more severe when the host's immune system is compromised. Rhoptry proteins are major virulence factors that enable to invade host cells.
View Article and Find Full Text PDFInt J Biol Sci
January 2025
Department of Urology, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
Clear cell renal cell carcinoma (ccRCC) is one of the most common and aggressive malignancies of the urinary system. Despite being the first-line treatment for advanced ccRCC, vascular endothelial growth factor receptor inhibitors (VEGFRis) face significant limitations due to both initial and acquired resistance, which impede complete tumor eradication. Using a CRISPR/Cas9 library screening approach, was identified as a resistance-associated gene for three prevalent VEGFRis (Sunitinib, Axitinib, and Sorafenib).
View Article and Find Full Text PDFCell Syst
December 2024
Center for Bioinformatics and Computational Medicine, Ann Arbor, MI 48109, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA; Program in Chemical Biology, University of Michigan, Ann Arbor, MI, USA; Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI 48109, USA. Electronic address:
While proliferating cells optimize their metabolism to produce biomass, the metabolic objectives of cells that perform non-proliferative tasks are unclear. The opposing requirements for optimizing each objective result in a trade-off that forces single cells to prioritize their metabolic needs and optimally allocate limited resources. Here, we present single-cell optimization objective and trade-off inference (SCOOTI), which infers metabolic objectives and trade-offs in biological systems by integrating bulk and single-cell omics data, using metabolic modeling and machine learning.
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