title: Designer Nuclease-Mediated Generation of Knockout THP1 Cells.
abstract: Recent developments in the field of designer nucleases allow the efficient and specific manipulation of genomic architectures in eukaryotic cell lines. To this end, it has become possible to introduce DNA double strand breaks (DSBs) at user-defined genomic loci. If located in critical coding regions of genes, thus induced DSBs can lead to insertions or deletions (indels) that result in frameshift mutations and thereby the knockout of the target gene. In this chapter, we describe a step-by-step workflow for establishing knockout cell clones of the difficult-to-transfect suspension cell line THP1. The here described protocol encompasses electroporation, cell cloning, and a deep sequencing-based genotyping step that allows the in-parallel analysis of 96 cell clones per gene of interest. Furthermore, we describe the use of the analysis tool OutKnocker that allows rapid identification of cell clones with all-allelic frameshift mutations.
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Straße 25, Bonn, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Schmidt
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Straße 25, Bonn, Germany.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Schmid-Burgk
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Straße 25, Bonn, Germany.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Ebert
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Straße 25, Bonn, Germany.
authors.forename: Moritz M
authors.initials: MM
authors.lastname: Gaidt
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Straße 25, Bonn, Germany. veit.hornung@uni-bonn.de.
authors.forename: Veit
authors.initials: V
authors.lastname: Hornung
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D004720:Endonucleases / Q000235:genetics*; D016368:Frameshift Mutation; D055786:Gene Knockout Techniques / Q000379:methods*; D016678:Genome; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D054643:INDEL Mutation
chemical_list: D004720:Endonucleases
keywords: Barcoded deep sequencing; CRISPR/Cas9; Genome editing; Genotyping; Knockout cell lines