a59b43c891087f1d3405f5a5bd6cb4cc genes_a2021v12n4p548.pdf c536fcf39e2dc0bd3c16d2193a5001f685291192 genes_a2021v12n4p548.pdf 42a45894f461eabbe7c488b36d2c4990b07455b35036529224f57b4160dd8066 genes_a2021v12n4p548.pdf Title: Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulates the Cellular Phenotypes of Fanconi Anemia Subject: Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore, better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they (i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, (ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore (iii) do not form FANCD2 nuclear foci, and (iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells. Keywords: Fanconi anemia; CRISPR/Cas9; gene editing; FANCA; head and neck cancer Author: Ricardo Errazquin, Esther Sieiro, Pilar Moreno, María José Ramirez, Corina Lorz, Jorge Peral, Jessica Ortiz, José Antonio Casado, Francisco J. Roman-Rodriguez, Helmut Hanenberg, Paula Río, Jordi Surralles, Carmen Segrelles and Ramon Garcia-Escudero Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Apr 12 03:33:36 2021 CEST ModDate: Mon Apr 12 04:13:06 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1812682 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- OPZDHV+VnURWPalladioL Type 1 Custom yes yes no 10 0 EJRQNW+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 FYTDJI+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 LDGXFE+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 YSPQJI+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 KONDUX+EuphemiaUCAS-Bold Type 1C WinAnsi yes yes no 36 0 HIZBRM+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 39 0 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TrueType Identity-H yes yes yes 157 0 CCEDFA+PalatinoLinotype TrueType WinAnsi yes yes no 163 0 CCEDFC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 166 0 CAMXTK+EURM10 Type 1 Builtin yes yes no 173 0 CCEDEO+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 180 0 CCEDEP+PalatinoLinotype CID TrueType Identity-H yes yes yes 183 0 CCEDFA+PalatinoLinotype TrueType WinAnsi yes yes no 189 0 CCEDFC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 192 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-02-01 02:32:02 CET RepresentationInformation: genes_a2021v12n4p548.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2021-12-17 11:34:54 CET Size: 1812682 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 377 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Biallelic Mutation in the FANCA Gene by CRISPR/Cas9 Editing in Non-FA HNSCC Cell Lines Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: FANCA-Mutant Clones Are Hypersensitive to ICL-Agents Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: FANCA-Mutant Clones Are Defective in FANCD2 Monoubiquitination and Nuclear Foci Formation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Increased Chromosome Fragility and G2 Arrest in FANCA-Mutant Clones Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: FANCA-Mutation Does Not Affect Growth Rates in HNSCC Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: FANCA-Mutation Augments Cell Migration in HNSCC Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: Cell Culture and Genotoxic Agents Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Cell Line Nucleofection and CRISPR/Cas9 Editing within Exon 4 of FANCA Gene Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Protein Extraction and Western Blotting Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Mitomycin C (MMC) and Cisplatin Sensitivity Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: FANCD2 Immunofluorescence Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Chromosome Fragility and G2 Arrest by the Flow Cytometry Micronucleus (MN) Test Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Cell Growth Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Wound Healing Assay for Cell Migration Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Info: Title: Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulates the Cellular Phenotypes of Fanconi Anemia Author: Ricardo Errazquin, Esther Sieiro, Pilar Moreno, María José Ramirez, Corina Lorz, Jorge Peral, Jessica Ortiz, José Antonio Casado, Francisco J. Roman-Rodriguez, Helmut Hanenberg, Paula Río, Jordi Surralles, Carmen Segrelles and Ramon Garcia-Escudero Subject: Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore, better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they (i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, (ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore (iii) do not form FANCD2 nuclear foci, and (iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells. 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