title: TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice.
abstract: Over the last decades, much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology. The breakthrough has been made in recent years with the advent of sequence-specific endonucleases, especially zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) guided nucleases (e.g., Cas9). In higher eukaryotic organisms, site-directed mutagenesis usually can be achieved through non-homologous end-joining (NHEJ) repair to the DNA double-strand breaks (DSBs) caused by the exogenously applied nucleases. However, site-specific gene replacement or genuine genome editing through homologous recombination (HR) repair to DSBs remains a challenge. As a proof of concept gene replacement through TALEN-based HR in rice (Oryza sativa), we successfully produced double point mutations in rice acetolactate synthase gene (OsALS) and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations. After ballistic delivery into rice calli of TALEN construct and donor DNA, nine HR events with different genotypes of OsALS were obtained in T0 generation at the efficiency of 1.4%-6.3% from three experiments. The HR-mediated gene edits were heritable to the progeny of T1 generation. The edited T1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance. The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.
authors.affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames 50011, USA.
authors.forename: Ting
authors.initials: T
authors.lastname: Li
authors.affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames 50011, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames 50011, USA.
authors.forename: Chih Ying
authors.initials: CY
authors.lastname: Chen
authors.affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames 50011, USA. Electronic address: byang@iastate.edu.
authors.forename: Bing
authors.initials: B
authors.lastname: Yang
mesh_terms: D000095:Acetolactate Synthase / Q000235:genetics; D001565:Benzoates / Q000494:pharmacology; D002934:Cinnamates / Q000378:metabolism; D018744:DNA, Plant / Q000235:genetics*; D003851:Deoxyribonucleases / Q000378:metabolism*; D004351:Drug Resistance / Q000235:genetics*; D000072669:Gene Editing; D006540:Herbicides / Q000494:pharmacology*; D059765:Homologous Recombination / Q000235:genetics*; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D012275:Oryza / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D030821:Plants, Genetically Modified; D017354:Point Mutation; D011743:Pyrimidines / Q000494:pharmacology
chemical_list: D001565:Benzoates; D002934:Cinnamates; D018744:DNA, Plant; D006540:Herbicides; D011743:Pyrimidines; D006921:Hygromycin B; C026273:hygromycin A; C549011:bispyribac; D000095:Acetolactate Synthase; D003851:Deoxyribonucleases
keywords: Acetolactate synthase; Gene replacement; Genome editing; Herbicide resistance; Homologous recombination; Rice; Site-directed mutagenesis; TALEN