{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,25]],"date-time":"2026-03-25T23:58:47Z","timestamp":1774483127337,"version":"3.50.1"},"reference-count":41,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2022,12,28]],"date-time":"2022-12-28T00:00:00Z","timestamp":1672185600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Many variants of uncertain significance (VUS) have been detected in clinical cancer cases using next-generation sequencing-based cancer gene panel analysis. One strategy for the elucidation of VUS is the functional analysis of cultured cancer cell lines that harbor targeted gene variants using genome editing. Genome editing is a powerful tool for creating desired gene alterations in cultured cancer cell lines. However, the efficiency of genome editing varies substantially among cell lines of interest. We performed comparative studies to determine the optimal editing conditions for the introduction of platelet-derived growth factor receptor alpha (PDGFRA) variants in human glioblastoma multiforme (GBM) cell lines. After monitoring the copy numbers of PDGFRA and the expression level of the PDGFR\u03b1 protein, four GBM cell lines (U-251 MG, KNS-42, SF126, and YKG-1 cells) were selected for the study. To compare the editing efficiency in these GBM cell lines, the modes of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) delivery (plasmid vs. ribonucleoprotein (RNP)), methods of transfection (lipofection vs. electroporation), and usefulness of cell sorting were then evaluated. Herein, we demonstrated that electroporation-mediated transfer of Cas9 with single-guide RNA (Cas9 RNP complex) could sufficiently edit a target nucleotide substitution, irrespective of cell sorting. As the Cas9 RNP complex method showed a higher editing efficiency than the Cas9 plasmid lipofection method, it was the optimal method for single-nucleotide editing in human GBM cell lines under our experimental conditions.<\/jats:p>","DOI":"10.3390\/ijms24010500","type":"journal-article","created":{"date-parts":[[2022,12,28]],"date-time":"2022-12-28T08:38:19Z","timestamp":1672216699000},"page":"500","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Genome Editing Using Cas9 Ribonucleoprotein Is Effective for Introducing PDGFRA Variant in Cultured Human Glioblastoma Cell Lines"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7580-7613","authenticated-orcid":false,"given":"Taiji","family":"Hamada","sequence":"first","affiliation":[{"name":"Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Seiya","family":"Yokoyama","sequence":"additional","affiliation":[{"name":"Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Toshiaki","family":"Akahane","sequence":"additional","affiliation":[{"name":"Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kei","family":"Matsuo","sequence":"additional","affiliation":[{"name":"Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7508-1624","authenticated-orcid":false,"given":"Akihide","family":"Tanimoto","sequence":"additional","affiliation":[{"name":"Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,28]]},"reference":[{"key":"ref_1","first-page":"00011","article-title":"OncoKB: A Precision Oncology Knowledge Base","volume":"17","author":"Chakravarty","year":"2017","journal-title":"JCO Precis Oncol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1614","DOI":"10.1002\/humu.23645","article-title":"ClinGen Variant Curation Expert Panel experiences and standardized processes for disease and gene-level specification of the ACMG\/AMP guidelines for sequence variant interpretation","volume":"39","author":"Milko","year":"2018","journal-title":"Hum. 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