title: An efficient TALEN mutagenesis system in rice.
abstract: Targeted gene mutagenesis is a powerful tool for elucidating gene function and facilitating genetic improvement in rice. TALENs (transcription activator-like effector nucleases), consisting of a custom TALE DNA binding domain fused to a nonspecific FokI cleavage domain, are one of the most efficient genome engineering methods developed to date. The technology of TALENs allows DNA double-strand breaks (DSBs) to be introduced into predetermined chromosomal loci. DSBs trigger DNA repair mechanisms and can result in loss of gene function by error-prone non-homologous end joining (NHEJ), or they can be exploited to modify gene function or activity by precise homologous recombination (HR). In this paper, we describe a detailed protocol for constructing TALEN expression vectors, assessing nuclease activities in vivo using rice protoplast-based assays, generating and introducing TALEN DNAs into embryogenic calluses of rice and identifying TALEN-generated mutations at targeted genomic sites. Using these methods, T0 rice plants resulting from TALEN mutagenesis can be produced within 4-5 months.
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Kunling
authors.initials: K
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Shan
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: cxgao@genetics.ac.cn.
authors.forename: Caixia
authors.initials: C
authors.lastname: Gao
mesh_terms: D053903:DNA Breaks, Double-Stranded; D003851:Deoxyribonucleases / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D012275:Oryza / Q000235:genetics*; D011523:Protoplasts
chemical_list: D003851:Deoxyribonucleases
keywords: Gene knockout; Rice; TALENs; Targeted mutagenesis