title: Footprintless disruption of prosurvival genes in aneuploid cancer cells using CRISPR/Cas9 technology.
abstract: CRISPR/Cas9 has emerged as a powerful methodology for the targeted editing of genomic DNA sequences. Nevertheless, the intrinsic inefficiency of transfection methods required to use this technique with cultured cells requires the selection and isolation of successfully modified cells, which invariably subjects the cells to stress. Here we report a workflow that allows the isolation of genomically modified cells, even where loss of functional alleles constitutes a selective disadvantage owing to impaired ability to survive stress. Using targeted disruption of the Id1 and Id3 genes in murine B16-F10 and Ret melanoma cell lines as an example, we show that the method allows for the footprintless isolation of CRISPR/Cas9-modified aneuploid cancer cells. We also provide evidence that serial CRISPR/Cas9 modifications can occur, for example when initial homologous recombination events introduce cryptic PAM sequences, and demonstrate that multiple alleles can be successfully targeted in aneuploid cancer cells. By sequencing individual alleles we also found evidence for CRISPR/Cas9-induced transposable element insertion, albeit at a low frequency. This workflow should have broad application in the functional analysis of prosurvival gene function in cultured cells.
authors.affiliation: a Centre for Biomedicine and Medical Technology Mannheim, Medical Faculty Mannheim, University Heidelberg, 68167 Mannheim, Germany.
authors.forename: Justyna M
authors.initials: JM
authors.lastname: Krachulec
authors.affiliation: a Centre for Biomedicine and Medical Technology Mannheim, Medical Faculty Mannheim, University Heidelberg, 68167 Mannheim, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Sedlmeier
authors.affiliation: a Centre for Biomedicine and Medical Technology Mannheim, Medical Faculty Mannheim, University Heidelberg, 68167 Mannheim, Germany.|b Institute for Toxicology and Genetics, Karlsruhe Institute of Technology Campus Nord, 76021 Karlsruhe, Germany.
authors.forename: Wilko
authors.initials: W
authors.lastname: Thiele
authors.affiliation: a Centre for Biomedicine and Medical Technology Mannheim, Medical Faculty Mannheim, University Heidelberg, 68167 Mannheim, Germany.|b Institute for Toxicology and Genetics, Karlsruhe Institute of Technology Campus Nord, 76021 Karlsruhe, Germany.
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Sleeman
mesh_terms: D000782:Aneuploidy; D000818:Animals; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D051798:Inhibitor of Differentiation Protein 1 / Q000235:genetics*; D051796:Inhibitor of Differentiation Proteins / Q000235:genetics*; D008546:Melanoma, Experimental / Q000235:genetics / Q000473:pathology; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods; D057188:Workflow
chemical_list: C497455:Idb1 protein, mouse; D051798:Inhibitor of Differentiation Protein 1; D051796:Inhibitor of Differentiation Proteins; C067679:Idb3 protein, mouse
keywords: B16-F10; CRISPR/Cas9; Id genes; Ret melanoma cells; gènes Id; mélanomes Ret