title: CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants.
abstract: Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3) and protoporphyrinogen oxidase (PPO) genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80), b-NHEJ (parp1 parp2), or both (ku80 parp1 parp2). We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants.
authors.affiliation: Department of Molecular and Developmental Genetics, Institute of Biology, Leiden University, 2333 BE, The Netherlands.
authors.forename: Hexi
authors.initials: H
authors.lastname: Shen
authors.affiliation: Department of Molecular and Developmental Genetics, Institute of Biology, Leiden University, 2333 BE, The Netherlands.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Strunks
authors.affiliation: Department of Molecular and Developmental Genetics, Institute of Biology, Leiden University, 2333 BE, The Netherlands.
authors.forename: Bart J P M
authors.initials: BJ
authors.lastname: Klemann
authors.affiliation: Department of Molecular and Developmental Genetics, Institute of Biology, Leiden University, 2333 BE, The Netherlands.
authors.forename: Paul J J
authors.initials: PJ
authors.lastname: Hooykaas
authors.affiliation: Department of Molecular and Developmental Genetics, Institute of Biology, Leiden University, 2333 BE, The Netherlands b.s.de.pater@biology.leidenuniv.nl.
authors.forename: Sylvia
authors.identifier: 0000-0003-2626-3661
authors.initials: S
authors.lastname: de Pater
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000235:genetics; D004265:DNA Helicases / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D005916:Globulins / Q000235:genetics; D059765:Homologous Recombination / Q000235:genetics*; D009154:Mutation; D000071137:Poly (ADP-Ribose) Polymerase-1 / Q000235:genetics*; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics*; D050768:Protoporphyrinogen Oxidase / Q000235:genetics; D055314:Seed Storage Proteins / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; D005916:Globulins; D055314:Seed Storage Proteins; C506891:cruciferin C, Arabidopsis; D050768:Protoporphyrinogen Oxidase; C000625018:AT4G02390 protein, Arabidopsis; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D004265:DNA Helicases; C469228:Ku80 protein, Arabidopsis
keywords: Arabidopsis thaliana; CRISPR/Cas9; KU80; double-strand break; nonhomologous end-joining