title: Gene editing by co-transformation of TALEN and chimeric RNA/DNA oligonucleotides on the rice OsEPSPS gene and the inheritance of mutations.
abstract: Although several site-speciﬁc nucleases (SSNs), such as zinc-ﬁnger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas, have emerged as powerful tools for targeted gene editing in many organisms, to date, gene targeting (GT) in plants remains a formidable challenge. In the present study, we attempted to substitute a single base in situ on the rice OsEPSPS gene by co-transformation of TALEN with chimeric RNA/DNA oligonucleotides (COs), including different strand composition such as RNA/DNA (C1) or DNA/RNA (C2) but contained the same target base to be substituted. In contrast to zero GT event obtained by the co-transformation of TALEN with homologous recombination plasmid (HRP), we obtained one mutant showing target base substitution although accompanied by undesired deletion of 12 bases downstream the target site from the co-transformation of TALEN and C1. In addition to this typical event, we also obtained 16 mutants with different length of base deletions around the target site among 105 calli lines derived from transformation of TALEN alone (4/19) as well as co-transformation of TELAN with either HRP (5/30) or C1 (2/25) or C2 (5/31). Further analysis demonstrated that the homozygous gene-edited mutants without foreign gene insertion could be obtained in one generation. The induced mutations in transgenic generation were also capable to pass to the next generation stably. However, the genotypes of mutants did not segregate normally in T1 population, probably due to lethal mutations. Phenotypic assessments in T1 generation showed that the heterozygous plants with either one or three bases deletion on target sequence, called d1 and d3, were more sensitive to glyphosate and the heterozygous d1 plants had significantly lower seed-setting rate than wild-type.
authors: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang Province, China.
authors: Mugui
authors: M
authors: Wang
authors: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang Province, China.
authors: Yujun
authors: Y
authors: Liu
authors: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang Province, China.
authors: Cuicui
authors: C
authors: Zhang
authors: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang Province, China.
authors: Jianping
authors: J
authors: Liu
authors: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang Province, China.
authors: Xin
authors: X
authors: Liu
authors: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang Province, China.
authors: Liangchao
authors: L
authors: Wang
authors: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang Province, China.
authors: Wenyi
authors: W
authors: Wang
authors: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang Province, China.
authors: Hao
authors: H
authors: Chen
authors: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang Province, China.
authors: Chuchu
authors: C
authors: Wei
authors: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang Province, China.
authors: Xiufen
authors: X
authors: Ye
authors: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang Province, China.
authors: Xinyuan
authors: X
authors: Li
authors: Institute of Crop Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang Province, China.
authors: Jumin
authors: J
authors: Tu
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D059765:Homologous Recombination; D040582:Inheritance Patterns; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D012275:Oryza / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D004720:Endonucleases; D005998:Glycine; D009841:Oligonucleotides; D010940:Plant Proteins; D000097797:Glyphosate