CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants
Autor: | Paul J. J. Hooykaas, Gary D Strunks, Hexi Shen, Sylvia de Pater, Bart J. P. M. Klemann |
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Jazyk: | angličtina |
Rok vydání: | 2017 |
Předmět: |
0301 basic medicine
double-strand break DNA End-Joining Repair Ku80 DNA Repair Arabidopsis thaliana Arabidopsis Poly (ADP-Ribose) Polymerase-1 nonhomologous end-joining Investigations QH426-470 03 medical and health sciences PARP1 Genetics CRISPR DNA Breaks Double-Stranded Protoporphyrinogen Oxidase Homologous Recombination Molecular Biology CRISPR/Cas9 Genetics (clinical) Polymerase KU80 biology Arabidopsis Proteins Cas9 Seed Storage Proteins fungi DNA Helicases Globulins Double Strand Break Repair DNA-Binding Proteins Non-homologous end joining enzymes and coenzymes (carbohydrates) 030104 developmental biology Mutation embryonic structures biology.protein CRISPR-Cas Systems Poly(ADP-ribose) Polymerases Homologous recombination |
Zdroj: | G3 : Genes, Genomes, Genetics, 7(1), 193-202 G3 : Genes, Genomes, Genetics G3: Genes, Genomes, Genetics, Vol 7, Iss 1, Pp 193-202 (2017) G3: Genes|Genomes|Genetics |
Popis: | 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. |
Databáze: | OpenAIRE |
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