Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes

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...

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Published in	Nature communications Vol. 8; no. 1; p. 14261
Main Authors	Liang, Zhen, Chen, Kunling, Li, Tingdong, Zhang, Yi, Wang, Yanpeng, Zhao, Qian, Liu, Jinxing, Zhang, Huawei, Liu, Cuimin, Ran, Yidong, Gao, Caixia
Format	Journal Article
Language	English
Published	London Nature Publishing Group UK 18.01.2017 Nature Publishing Group Nature Portfolio
Subjects	13/106 38/109 38/22 38/70 38/77 42 42/41 631/208/2491/2046 631/337/4041/3196 631/61/447/2311 CRISPR CRISPR-Cas Systems Deoxyribonucleic acid DNA DNA, Plant - genetics DNA, Plant - metabolism Embryos Gene Editing Genetic testing Genetics Genome editing Genomes Humanities and Social Sciences multidisciplinary Mutagenesis Mutation Plant Breeding Ribonucleoproteins - genetics Ribonucleoproteins - metabolism Science Science (multidisciplinary) Triticum - genetics Triticum - metabolism Wheat Beijing China China
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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. Protocols for crop genome editing would ideally be quick, efficient and specific while avoiding integration of transgenes into the genome of edited plants. Here, Liang et al . show that CRISPR/Cas9 ribonucleoproteins can be used to generate genome edited wheat plants in as little as nine weeks.
AbstractList	Protocols for crop genome editing would ideally be quick, efficient and specific while avoiding integration of transgenes into the genome of edited plants. Here, Lianget al. show that CRISPR/Cas9 ribonucleoproteins can be used to generate genome edited wheat plants in as little as nine weeks. 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. 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. Protocols for crop genome editing would ideally be quick, efficient and specific while avoiding integration of transgenes into the genome of edited plants. Here, Liang et al . show that CRISPR/Cas9 ribonucleoproteins can be used to generate genome edited wheat plants in as little as nine weeks.
ArticleNumber	14261
Author	Liang, Zhen Zhao, Qian Zhang, Huawei Wang, Yanpeng Liu, Jinxing Ran, Yidong Chen, Kunling Zhang, Yi Liu, Cuimin Li, Tingdong Gao, Caixia
Author_xml	– sequence: 1 givenname: Zhen surname: Liang fullname: Liang, Zhen organization: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences – sequence: 2 givenname: Kunling surname: Chen fullname: Chen, Kunling organization: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences – sequence: 3 givenname: Tingdong surname: Li fullname: Li, Tingdong organization: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences – sequence: 4 givenname: Yi surname: Zhang fullname: Zhang, Yi organization: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences – sequence: 5 givenname: Yanpeng surname: Wang fullname: Wang, Yanpeng organization: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences – sequence: 6 givenname: Qian surname: Zhao fullname: Zhao, Qian organization: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences – sequence: 7 givenname: Jinxing surname: Liu fullname: Liu, Jinxing organization: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences – sequence: 8 givenname: Huawei surname: Zhang fullname: Zhang, Huawei organization: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences – sequence: 9 givenname: Cuimin orcidid: 0000-0003-4982-4804 surname: Liu fullname: Liu, Cuimin organization: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences – sequence: 10 givenname: Yidong surname: Ran fullname: Ran, Yidong organization: Genovo Biotechnology Co., Ltd – sequence: 11 givenname: Caixia surname: Gao fullname: Gao, Caixia email: cxgao@genetics.ac.cn organization: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences
BackLink	https://www.ncbi.nlm.nih.gov/pubmed/28098143$$D View this record in MEDLINE/PubMed
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Snippet	Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of... Protocols for crop genome editing would ideally be quick, efficient and specific while avoiding integration of transgenes into the genome of edited plants....
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SubjectTerms	13/106 38/109 38/22 38/70 38/77 42 42/41 631/208/2491/2046 631/337/4041/3196 631/61/447/2311 CRISPR CRISPR-Cas Systems Deoxyribonucleic acid DNA DNA, Plant - genetics DNA, Plant - metabolism Embryos Gene Editing Genetic testing Genetics Genome editing Genomes Humanities and Social Sciences multidisciplinary Mutagenesis Mutation Plant Breeding Ribonucleoproteins - genetics Ribonucleoproteins - metabolism Science Science (multidisciplinary) Triticum - genetics Triticum - metabolism Wheat
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Title	Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes
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