Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells

Geyer, Fabian; Geyer, Maximilian; Klapproth, Sarah; Wolff, Klaus‐Dietrich; Nieberler, Markus

doi:10.1016/j.xpro.2023.102366

Cited by 3 publications

(7 citation statements)

References 6 publications

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“…Initially, three different guide sequences were tested and the best performing CRISPR RNA (crRNA) was chosen. Editing efficiency was highly dependent on the transfected amount of RNP complexes and cell confluency, resulting in up to 73% (mean 67.33%) insertion and deletion mutations (INDEL), as we reported previously 32 . Given the high Indel%, a single crRNA approach with limiting dilution was possible.…”

Section: Resultssupporting

confidence: 70%

“…In a previous publication by us, we described the established workflow of CRISPR/Cas9-mediated knockdown of CHD4, utilizing lipofection of ribonucleoprotein complexes (RNP) 32 . Suitable custom guide sequences, targeting the human CHD4 locus on chromosome 12, were designed using the online tool CHOPCHOP ( https://chopchop.cbu.uib.no ) 56 and ordered as crRNA from IDT™ (IDT, Coralville, Iowa, USA).…”

Section: Methodsmentioning

confidence: 99%

“…The CHD4 alleles were amplified with a previously designed primer pair as mentioned above, receiving an amplicon length of 480 bp. For detailed PCR settings, please refer to Geyer et al 32 .…”

Section: Methodsmentioning

confidence: 99%

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CHD4 acts as a prognostic factor and drives radioresistance in HPV negative HNSCC

Geyer,

Reuning

et al. 2024

Sci Rep

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Despite great efforts in improving existing therapies, the outcome of patients with advanced radioresistant HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. The chromatin remodeler Chromodomain helicase DNA binding protein 4 (CHD4) is involved in different DNA-repair mechanisms, but the role and potential in HNSCC has not been explored yet. In the present study, we evaluated the prognostic significance of CHD4 expression using in silico analysis of the pan-cancer dataset. Furthermore, we established a monoclonal HNSCC CHD4 knockdown cell clone utilizing the CRISPR/Cas9 system. Effects of lower CHD4 expression on radiosensitivity after increasing doses of ionizing radiation were characterized using clonogenic assays and cell numbers. The in silico analysis revealed that high CHD4 expression is associated with significant poorer overall survival of HPV-negative HNSCC patients. Additionally, the knockdown of CHD4 significantly increased the radiosensitivity of HNSCC cells. Therefore, CHD4 might be involved in promoting radioresistance in hard-to-treat HPV-negative HNSCC entities. We conclude that CHD4 could serve as a prognostic factor in HPV-negative HNSCC tumors and is a potential target protein overcoming radioresistance in HNSCC. Our results and the newly established cell clone laid the foundation to further characterize the underlying mechanisms and ultimately use CHD4 in HNSCC therapies.

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Section: Resultssupporting

confidence: 70%

Section: Methodsmentioning

confidence: 99%

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CHD4 acts as a prognostic factor and drives radioresistance in HPV negative HNSCC

Geyer,

Reuning

et al. 2024

Sci Rep

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“…The derivation of the cell line from a metastatic cancer is of great importance in evaluating the impact of ITGB6 on the metastatic potential. The cells were maintained in Dulbecco´s Modified Eagle´s Medium (DMEM) (Sigma Aldrich, St. Louis, Missouri, USA) supplemented with 10% (v/v) fetal calf serum (FCS) (Sigma Aldrich, St. Louis, Missouri, USA) and cultured in accordance with previously described methodology [ 32 ].…”

Section: Methodsmentioning

confidence: 99%

“…For detailed information on the protocol, exact amounts of applied components and the entire workflow, please refer to Geyer et al [ 32 ].…”

Section: Methodsmentioning

confidence: 99%

CRISPR/Cas9-mediated knock out of ITGB6 in human OSCC cells reduced migration and proliferation ability

Geyer,

Reuning

et al. 2024

Head Face Med

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Background The treatment of oral squamous cell carcinoma (OSCC) remains challenging and survival rates have not been improved significantly over the past decades. Integrins have been recognized driving the cancer progression and high expression levels cause poor outcomes in patients afflicted with OSCC. Integrin αvβ6 and its subunit integrin beta 6 (ITGB6) were discovered to enhance the invasiveness by providing beneficial effects on downstream pathways promoting the cancer progression. The objective of this study was to establish a CRISPR/Cas9-mediated knock out of ITGB6 in the human OSCC cell line HN and investigate the effects on the migration and proliferation ability. Methods ITGB6 knock out was performed using the CRISPR/Cas9-system, RNPs, and lipofection. Monoclonal cell clones were achieved by limiting dilution and knock out verification was carried out by sanger sequencing and FACS on protein level. The effects of the knock out on the proliferation and migration ability were evaluated by using MTT and scratch assays. In addition, in silico TCGA analysis was utilized regarding the effects of ITGB6 on overall survival and perineural invasion. Results In silico analysis revealed a significant impact of ITGB6 mRNA expression levels on the overall survival of patients afflicted with OSCC. Additionally, a significantly higher rate of perineural invasion was discovered. CRISPR/Cas9-mediated knock out of ITGB6 was performed in the OSCC cell line HN, resulting in the generation of a monoclonal knock out clone. The knock out clone exhibited a significantly reduced migration and proliferation ability when compared to the wildtype. Conclusions ITGB6 is a relevant factor in the progression of OSCC and can be used for the development of novel treatment strategies. The present study is the first to establish a monoclonal CRISPR/Cas9-mediated ITGB6 knockout cell clone derived from an OSCC cell line. It suggests that ITGB6 has a significant impact on the proliferative and migratory capacity in vitro.

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Optimized CRISPR-based knockout in BeWo cells

Yin,

Esbin

2024

Placenta

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Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells

Cited by 3 publications

References 6 publications

CHD4 acts as a prognostic factor and drives radioresistance in HPV negative HNSCC

CHD4 acts as a prognostic factor and drives radioresistance in HPV negative HNSCC

CRISPR/Cas9-mediated knock out of ITGB6 in human OSCC cells reduced migration and proliferation ability

Optimized CRISPR-based knockout in BeWo cells

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