title: Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis.
abstract: This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.
authors.affiliation: Biotech Research and Innovation Centre (BRIC), Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Lindsey A
authors.initials: LA
authors.lastname: Lonowski
authors.affiliation: Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and Odontology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Narimatsu
authors.affiliation: Biotech Research and Innovation Centre (BRIC), Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Anjum
authors.initials: A
authors.lastname: Riaz
authors.affiliation: Biotech Research and Innovation Centre (BRIC), Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Delay
authors.affiliation: Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and Odontology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Zhang
authors.identifier: 0000-0002-4756-5813
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Biotech Research and Innovation Centre (BRIC), Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Francesco
authors.initials: F
authors.lastname: Niola
authors.affiliation: Biotech Research and Innovation Centre (BRIC), Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Duda
authors.affiliation: Danish Stem Cell Center (DanStem), University of Copenhagen, Copenhagen, Denmark.
authors.forename: Elke A
authors.initials: EA
authors.lastname: Ober
authors.affiliation: Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and Odontology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
authors.affiliation: Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and Odontology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Hans H
authors.initials: HH
authors.lastname: Wandall
authors.affiliation: GI Cell Biology Research Laboratory, Boston Children´s Hospital and Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Steen H
authors.initials: SH
authors.lastname: Hansen
authors.affiliation: Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and Odontology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Eric P
authors.initials: EP
authors.lastname: Bennett
authors.affiliation: Biotech Research and Innovation Centre (BRIC), Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Frödin
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004252:DNA Mutational Analysis / Q000379:methods*; D003851:Deoxyribonucleases / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D000072669:Gene Editing / Q000379:methods*; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D023281:Genomics / Q000379:methods*; D054643:INDEL Mutation
chemical_list: D003851:Deoxyribonucleases