title: Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes.
abstract: Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
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.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Tingdong
authors.initials: T
authors.lastname: Li
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Yanpeng
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhao
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: Jinxing
authors.initials: J
authors.lastname: Liu
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: Huawei
authors.initials: H
authors.lastname: Zhang
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: Cuimin
authors.identifier: 0000-0003-4982-4804
authors.initials: C
authors.lastname: Liu
authors.affiliation: Genovo Biotechnology Co., Ltd., Tianjin 301700, China.
authors.forename: Yidong
authors.initials: Y
authors.lastname: Ran
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: Caixia
authors.initials: C
authors.lastname: Gao
mesh_terms: D064113:CRISPR-Cas Systems; D018744:DNA, Plant / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing; D009154:Mutation; D000069600:Plant Breeding; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D014908:Triticum / Q000235:genetics* / Q000378:metabolism
chemical_list: D018744:DNA, Plant; D012261:Ribonucleoproteins