title: Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks.
abstract: The creation of a DNA break at a specific locus by a designer endonuclease can be harnessed to edit a genome. However, DNA breaks may engage one of several competing repair pathways that lead to distinct types of genomic alterations. Therefore, understanding the contribution of different repair pathways following the introduction of a targeted DNA break is essential to further advance the safety and efficiency of nuclease-induced genome modification. To gain insight into the role of different DNA repair pathways in resolving nuclease-induced DNA breaks into genome editing outcomes, we previously developed a fluorescent-based reporter system, designated the Traffic Light Reporter, which provides a readout of gene targeting and gene disruption downstream of a targeted DNA double-strand break. Here we describe two related but novel reporters that extend this technology: one that allows monitoring of the transcriptional activity at the reporter locus, and thus can be applied to interrogate break resolution at active and repressed loci; and a second that reads out single-strand annealing in addition to gene targeting and gene disruption. Application of these reporters to assess repair pathway usage in several common gene editing contexts confirms the importance that chromatin status and initiation of end resection have on the resolution of nuclease-induced breaks.
authors.affiliation: Center of Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, 98101 Washington, USA, Program in Molecular and Cellular Biology, University of Washington, Seattle, 98195 Washington, USA, Department of Immunology, University of Washington, Seattle, 98195 Washington, USA and Department of Biochemistry, University of Washington, Seattle, 98195 Washington, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Kuhar
authors.forename: Kamila S
authors.initials: KS
authors.lastname: Gwiazda
authors.forename: Olivier
authors.initials: O
authors.lastname: Humbert
authors.forename: Tyler
authors.initials: T
authors.lastname: Mandt
authors.forename: Joey
authors.initials: J
authors.lastname: Pangallo
authors.forename: Michelle
authors.initials: M
authors.lastname: Brault
authors.forename: Iram
authors.initials: I
authors.lastname: Khan
authors.forename: Nancy
authors.initials: N
authors.lastname: Maizels
authors.forename: David J
authors.initials: DJ
authors.lastname: Rawlings
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scharenberg
authors.forename: Michael T
authors.initials: MT
authors.lastname: Certo
mesh_terms: D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004706:Endodeoxyribonucleases; D005434:Flow Cytometry; D005453:Fluorescence; D020868:Gene Silencing; D005796:Genes; D017930:Genes, Reporter; D056426:Genetic Loci; D016678:Genome; D023281:Genomics / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D008164:Luminescent Proteins; D004706:Endodeoxyribonucleases