title: A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice.
abstract: We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing.
authors.affiliation: Plant Genome Engineering Research Unit (A.N.-Y., H.S., A.M., S.T.), Rice Applied Genomics Research Unit (T.H., K.S.), and Advanced Genomics Laboratory (M.H., Y.K.), National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (T.C., C.S., D.F.V.);Center for Collaboration among Agriculture, Industry, and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); andKihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Ayako
authors.identifier: http://orcid.org/0000-0003-2781-1359
authors.initials: A
authors.lastname: Nishizawa-Yokoi
authors.affiliation: Plant Genome Engineering Research Unit (A.N.-Y., H.S., A.M., S.T.), Rice Applied Genomics Research Unit (T.H., K.S.), and Advanced Genomics Laboratory (M.H., Y.K.), National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (T.C., C.S., D.F.V.);Center for Collaboration among Agriculture, Industry, and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); andKihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Tomas
authors.initials: T
authors.lastname: Cermak
authors.affiliation: Plant Genome Engineering Research Unit (A.N.-Y., H.S., A.M., S.T.), Rice Applied Genomics Research Unit (T.H., K.S.), and Advanced Genomics Laboratory (M.H., Y.K.), National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (T.C., C.S., D.F.V.);Center for Collaboration among Agriculture, Industry, and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); andKihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Tomoki
authors.identifier: http://orcid.org/0000-0001-5630-4685
authors.initials: T
authors.lastname: Hoshino
authors.affiliation: Plant Genome Engineering Research Unit (A.N.-Y., H.S., A.M., S.T.), Rice Applied Genomics Research Unit (T.H., K.S.), and Advanced Genomics Laboratory (M.H., Y.K.), National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (T.C., C.S., D.F.V.);Center for Collaboration among Agriculture, Industry, and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); andKihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Sugimoto
authors.affiliation: Plant Genome Engineering Research Unit (A.N.-Y., H.S., A.M., S.T.), Rice Applied Genomics Research Unit (T.H., K.S.), and Advanced Genomics Laboratory (M.H., Y.K.), National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (T.C., C.S., D.F.V.);Center for Collaboration among Agriculture, Industry, and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); andKihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Saika
authors.affiliation: Plant Genome Engineering Research Unit (A.N.-Y., H.S., A.M., S.T.), Rice Applied Genomics Research Unit (T.H., K.S.), and Advanced Genomics Laboratory (M.H., Y.K.), National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (T.C., C.S., D.F.V.);Center for Collaboration among Agriculture, Industry, and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); andKihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Akiko
authors.initials: A
authors.lastname: Mori
authors.affiliation: Plant Genome Engineering Research Unit (A.N.-Y., H.S., A.M., S.T.), Rice Applied Genomics Research Unit (T.H., K.S.), and Advanced Genomics Laboratory (M.H., Y.K.), National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (T.C., C.S., D.F.V.);Center for Collaboration among Agriculture, Industry, and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); andKihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Keishi
authors.initials: K
authors.lastname: Osakabe
authors.affiliation: Plant Genome Engineering Research Unit (A.N.-Y., H.S., A.M., S.T.), Rice Applied Genomics Research Unit (T.H., K.S.), and Advanced Genomics Laboratory (M.H., Y.K.), National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (T.C., C.S., D.F.V.);Center for Collaboration among Agriculture, Industry, and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); andKihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Masao
authors.initials: M
authors.lastname: Hamada
authors.affiliation: Plant Genome Engineering Research Unit (A.N.-Y., H.S., A.M., S.T.), Rice Applied Genomics Research Unit (T.H., K.S.), and Advanced Genomics Laboratory (M.H., Y.K.), National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (T.C., C.S., D.F.V.);Center for Collaboration among Agriculture, Industry, and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); andKihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Katayose
authors.affiliation: Plant Genome Engineering Research Unit (A.N.-Y., H.S., A.M., S.T.), Rice Applied Genomics Research Unit (T.H., K.S.), and Advanced Genomics Laboratory (M.H., Y.K.), National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (T.C., C.S., D.F.V.);Center for Collaboration among Agriculture, Industry, and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); andKihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Colby
authors.identifier: http://orcid.org/0000-0002-6774-7227
authors.initials: C
authors.lastname: Starker
authors.affiliation: Plant Genome Engineering Research Unit (A.N.-Y., H.S., A.M., S.T.), Rice Applied Genomics Research Unit (T.H., K.S.), and Advanced Genomics Laboratory (M.H., Y.K.), National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (T.C., C.S., D.F.V.);Center for Collaboration among Agriculture, Industry, and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); andKihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Voytas
authors.affiliation: Plant Genome Engineering Research Unit (A.N.-Y., H.S., A.M., S.T.), Rice Applied Genomics Research Unit (T.H., K.S.), and Advanced Genomics Laboratory (M.H., Y.K.), National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (T.C., C.S., D.F.V.);Center for Collaboration among Agriculture, Industry, and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); andKihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.) stoki@affrc.go.jp.
authors.forename: Seiichi
authors.identifier: http://orcid.org/0000-0003-0066-6446
authors.initials: S
authors.lastname: Toki
mesh_terms: D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D018744:DNA, Plant / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D000069896:Transcription Activator-Like Effector Nucleases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018744:DNA, Plant; D010940:Plant Proteins; D000069896:Transcription Activator-Like Effector Nucleases; D011088:DNA Ligases