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Research Infrastructures of Strategic Relevance","award":["PTDC\/MEC-ONC\/31520\/2017"],"award-info":[{"award-number":["PTDC\/MEC-ONC\/31520\/2017"]}]},{"name":"Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance","award":["POCI-01-0145-FEDER-028779"],"award-info":[{"award-number":["POCI-01-0145-FEDER-028779"]}]},{"name":"Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance","award":["PTDC\/BIA-MIC\/28779\/2017"],"award-info":[{"award-number":["PTDC\/BIA-MIC\/28779\/2017"]}]},{"name":"Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance","award":["UID\/BIM\/04293\/2019"],"award-info":[{"award-number":["UID\/BIM\/04293\/2019"]}]},{"name":"Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic 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relevance in cancer biology is still unclear. Recent advances in gene editing techniques, particularly CRISPR\/Cas9 systems, offer new opportunities to evaluate the function of large genes, such as LRP1B. Using a dual sgRNA CRISPR\/Cas9 gene editing approach, this study aimed to assess the impact of disrupting LRP1B in glioblastoma cell biology. Four sgRNAs were designed for the dual targeting of two LRP1B exons (1 and 85). The U87 glioblastoma (GB) cell line was transfected with CRISPR\/Cas9 PX459 vectors. To assess LRP1B-gene-induced alterations and expression, PCR, Sanger DNA sequencing, and qRT-PCR were carried out. Three clones (clones B9, E6, and H7) were further evaluated. All clones presented altered cellular morphology, increased cellular and nuclear size, and changes in ploidy. Two clones (E6 and H7) showed a significant decrease in cell growth, both in vitro and in the in vivo CAM assay. Proteomic analysis of the clones\u2019 secretome identified differentially expressed proteins that had not been previously associated with LRP1B alterations. This study demonstrates that the dual sgRNA CRISPR\/Cas9 strategy can effectively edit LRP1B in GB cells, providing new insights into the impact of LRP1B deletions in GBM biology.<\/jats:p>","DOI":"10.3390\/ijms241411285","type":"journal-article","created":{"date-parts":[[2023,7,11]],"date-time":"2023-07-11T01:40:02Z","timestamp":1689039602000},"page":"11285","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Using a Dual CRISPR\/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9469-5680","authenticated-orcid":false,"given":"Joana","family":"Peixoto","sequence":"first","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Cancer Signaling and Metabolism Group, IPATIMUP\u2014Institute of Molecular Pathology and Immunology of the University of Porto, Rua Alfredo Allen 208, 4169-007 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1300-4006","authenticated-orcid":false,"given":"Catarina","family":"Pr\u00edncipe","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Cancer Signaling and Metabolism Group, IPATIMUP\u2014Institute of Molecular Pathology and Immunology of the University of Porto, Rua Alfredo Allen 208, 4169-007 Porto, Portugal"},{"name":"Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal"}]},{"given":"Ana","family":"Pestana","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Cancer Signaling and Metabolism Group, IPATIMUP\u2014Institute of Molecular Pathology and Immunology of the University of Porto, Rua Alfredo Allen 208, 4169-007 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6362-8255","authenticated-orcid":false,"given":"Hugo","family":"Os\u00f3rio","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"IPATIMUP\u2014Institute of Molecular Pathology and Immunology of the University of Porto, 4200-135 Porto, Portugal"},{"name":"FMUP\u2014Department of Pathology, Faculty of Medicine, University of Porto, Alameda Prof. Hern\u00e2ni Monteiro, 4200-319 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8521-2904","authenticated-orcid":false,"given":"Marta Teixeira","family":"Pinto","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"IPATIMUP\u2014Institute of Molecular Pathology and Immunology of the University of Porto, 4200-135 Porto, Portugal"}]},{"given":"Hugo","family":"Prazeres","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"IPATIMUP\u2014Institute of Molecular Pathology and Immunology of the University of Porto, 4200-135 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9607-6998","authenticated-orcid":false,"given":"Paula","family":"Soares","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Cancer Signaling and Metabolism Group, IPATIMUP\u2014Institute of Molecular Pathology and Immunology of the University of Porto, Rua Alfredo Allen 208, 4169-007 Porto, Portugal"},{"name":"IPATIMUP\u2014Institute of Molecular Pathology and Immunology of the University of Porto, 4200-135 Porto, Portugal"},{"name":"FMUP\u2014Department of Pathology, Faculty of Medicine, University of Porto, Alameda Prof. Hern\u00e2ni Monteiro, 4200-319 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5838-9659","authenticated-orcid":false,"given":"Raquel T.","family":"Lima","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"Cancer Signaling and Metabolism Group, IPATIMUP\u2014Institute of Molecular Pathology and Immunology of the University of Porto, Rua Alfredo Allen 208, 4169-007 Porto, Portugal"},{"name":"IPATIMUP\u2014Institute of Molecular Pathology and Immunology of the University of Porto, 4200-135 Porto, Portugal"},{"name":"FMUP\u2014Department of Pathology, Faculty of Medicine, University of Porto, Alameda Prof. Hern\u00e2ni Monteiro, 4200-319 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2023,7,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1843","DOI":"10.2147\/CMAR.S186142","article-title":"The unique invasiveness of glioblastoma and possible drug targets on extracellular matrix","volume":"11","author":"Hatoum","year":"2019","journal-title":"Cancer Manag. 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