Transcription activator-like effector nucleases （TALENs） and clustered regularly interspaced short palindromic repeats （CRISPR）/ CRISPR-associated （Cas） systems have emerged as powerful tools for genome editing ...Transcription activator-like effector nucleases （TALENs） and clustered regularly interspaced short palindromic repeats （CRISPR）/ CRISPR-associated （Cas） systems have emerged as powerful tools for genome editing in a variety of species. Here, we report, for the first time, targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, ZmlPKIA, ZmlPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic mutations. Also, we constructed two gRNAs targeting the ZmlPK gene in maize protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition, the CRISPR/Cas system induced targeted mutations in Z. mays protoplasts with efficiencies （13.1%） similar to those obtained with TALENs （9.1%）. Our results show that both TALENs and the CRISPR/Cas system can be used for genome modification in maize.展开更多
Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions, or for analyzing epistatic re- lationships in genetic pathways. Using ...Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions, or for analyzing epistatic re- lationships in genetic pathways. Using conventional methods, mutants with multiple gene mutations are generated by several rounds of intercrossing plants carrying a single mutation and identification of the offspring. This process is both timeconsuming and labor-intensive. Moreover, if the genes of interest are closely linked, multiple mutations can not be generated （Wijnker and de Jong, 2008）.展开更多
Tomato （Solanum lycopersicum） is the leading vegetable crop worldwide and an essential component of a healthy diet （Lin et al., 2014; Du et al., 2017）. Fruit color is regarded as one of the most important commerci...Tomato （Solanum lycopersicum） is the leading vegetable crop worldwide and an essential component of a healthy diet （Lin et al., 2014; Du et al., 2017）. Fruit color is regarded as one of the most important commercial traits in tomato （The Tomato Genome Consortium, 2012）. Consumers in different regions have different color preferences. For example, European and American consumers prefer red tomatoes, while pink tomatoes are more pop- ular in Asia countries, particularly in China and Japan （Ballester et al., 2010; Lin et al., 2014）. However, most of tomato breeding ma- terials are red-fruited, thus the generation of pink-fruited materials is very important for Asian tomato production. Metabolomics and genetics studies demonstrate that the pink trait results from the absence of yellow-colored flavonoid naringenin chalcone （NarCh） in the peels,展开更多
High grain protein content(GPC) reduces rice eating and cooking quality(ECQ). We generated OsAAP6 and OsAAP10 knockout mutants in three high-yielding japonica varieties and one japonica line using the CRISPR/Cas9 syst...High grain protein content(GPC) reduces rice eating and cooking quality(ECQ). We generated OsAAP6 and OsAAP10 knockout mutants in three high-yielding japonica varieties and one japonica line using the CRISPR/Cas9 system. Mutation efficiency varied with genetic background in the T_0 generation, and GPC in the T_1 generation decreased significantly,owing mainly to a reduction in glutelin content. Amylose content was down-regulated significantly in some Osaap6 and all Osaap10 mutants. The increased taste value of these mutants was supported by Rapid Visco Analysis(RVA) profiles, which showed higher peak viscosity and breakdown viscosity and lower setback viscosity than the wild type. There were no significant deficiencies in agronomic traits of the mutants. Targeted mutagenesis of OsAAP6 and OsAAP10, especially OsAAP10, using the CRISPR/Cas9 system can rapidly reduce GPC and improve ECQ of rice, providing a new strategy for the breeding cultivars with desired ECQ.展开更多
基因组定点编辑技术是研究基因功能和生物体改造的重要工具。CRISPR-Cas(Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins)系统是近年来发展的一种新型基因组编辑技术,该技术通过一段向导RN...基因组定点编辑技术是研究基因功能和生物体改造的重要工具。CRISPR-Cas(Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins)系统是近年来发展的一种新型基因组编辑技术,该技术通过一段向导RNA和配套的核酸酶就可对特定的基因组序列进行定点编辑,具有简单高效、应用广泛的特点,受到了生物学家的广泛关注。本文着重介绍CRISPR-Cas系统在植物中的研究进展,包括CRISPR-Cas9系统在植物中的应用与完善、扩大基因组编辑范围的研究、Cas9切口酶和失活酶的拓展、特异性单碱基突变编辑系统的研究、无外源DNA污染的植物基因编辑技术的发展以及基因组编辑技术在作物育种上的应用等方面。同时也提出了还需解决的问题,并展望了基因组编辑系统在作物育种中的应用前景,为开展这一领域的研究工作提供参考。展开更多
The CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPRassociated proteins) system was first identified in bacteria and archaea and can degrade exogenous substrates. It was developed as a gene ...The CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPRassociated proteins) system was first identified in bacteria and archaea and can degrade exogenous substrates. It was developed as a gene editing technology in 2013. Over the subsequent years, it has received extensive attention owing to its easy manipulation, high efficiency, and wide application in gene mutation and transcriptional regulation in mammals and plants. The process of CRISPR/Cas is optimized constantly and its application has also expanded dramatically. Therefore, CRISPR/Cas is considered a revolutionary technology in plant biology. Here, we introduce the mechanism of the type II CRISPR/Cas called CRISPR/Cas9, update its recent advances in various applications in plants, and discuss its future prospects to provide an argument for its use in the study of medicinal plants.展开更多
近年来,CRISPR-Cas系统病原核酸检测技术凭借其靶向精准、灵敏度高以及多重检测等特点,在即时检测(Point of Care testing,POCT)领域受到广泛关注。多种Cas蛋白具有顺式基因编辑功能和非特异性反式切割活性,基于这种特性成功实现了病原...近年来,CRISPR-Cas系统病原核酸检测技术凭借其靶向精准、灵敏度高以及多重检测等特点,在即时检测(Point of Care testing,POCT)领域受到广泛关注。多种Cas蛋白具有顺式基因编辑功能和非特异性反式切割活性,基于这种特性成功实现了病原核酸检测,极大改善了POCT产品灵敏度低的问题。其中,以Cas12为靶点并反式切割单链DNA探针的系统既简单又高效。因此,就CRISPR-Cas12a的历史、分类、检测原理和在核酸检测中的应用进展、开发检测试剂的困难等方面做一综述。展开更多
基金supported by the National Natural Science Foundation of China (Grant Nos. 31271795 and 31200273)
文摘Transcription activator-like effector nucleases （TALENs） and clustered regularly interspaced short palindromic repeats （CRISPR）/ CRISPR-associated （Cas） systems have emerged as powerful tools for genome editing in a variety of species. Here, we report, for the first time, targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, ZmlPKIA, ZmlPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic mutations. Also, we constructed two gRNAs targeting the ZmlPK gene in maize protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition, the CRISPR/Cas system induced targeted mutations in Z. mays protoplasts with efficiencies （13.1%） similar to those obtained with TALENs （9.1%）. Our results show that both TALENs and the CRISPR/Cas system can be used for genome modification in maize.
基金supported by the Agricultural Science and Technology Innovation Program
文摘Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions, or for analyzing epistatic re- lationships in genetic pathways. Using conventional methods, mutants with multiple gene mutations are generated by several rounds of intercrossing plants carrying a single mutation and identification of the offspring. This process is both timeconsuming and labor-intensive. Moreover, if the genes of interest are closely linked, multiple mutations can not be generated （Wijnker and de Jong, 2008）.
基金supported by the National Key Research and Development Program of China (2016YFD0100500 and 2016YFD0101703)the National Natural Science Foundation of China (Nos. 31601759 and 31471881)+1 种基金the Ministry of Agriculture of China (2016ZX08009-003-001)the Tai-Shan Scholar Program from the Shandong Provincial Government
文摘Tomato （Solanum lycopersicum） is the leading vegetable crop worldwide and an essential component of a healthy diet （Lin et al., 2014; Du et al., 2017）. Fruit color is regarded as one of the most important commercial traits in tomato （The Tomato Genome Consortium, 2012）. Consumers in different regions have different color preferences. For example, European and American consumers prefer red tomatoes, while pink tomatoes are more pop- ular in Asia countries, particularly in China and Japan （Ballester et al., 2010; Lin et al., 2014）. However, most of tomato breeding ma- terials are red-fruited, thus the generation of pink-fruited materials is very important for Asian tomato production. Metabolomics and genetics studies demonstrate that the pink trait results from the absence of yellow-colored flavonoid naringenin chalcone （NarCh） in the peels,
基金financially supported by National Key Research and Development Program of China(2016YFD0100501)the National Natural Science Foundation of China(31871241,31371233)+3 种基金the Natural Science Foundation of Jiangsu Province(BE2017345,PZCZ201702,BE2018351)the Research and Innovation Program of Postgraduate in Jiangsu Province(KYCX17_1886)the Priority Academic Program Development of Jiangsu Higher Education Institutionsthe Yangzhou University International Academic Exchange Fund。
文摘High grain protein content(GPC) reduces rice eating and cooking quality(ECQ). We generated OsAAP6 and OsAAP10 knockout mutants in three high-yielding japonica varieties and one japonica line using the CRISPR/Cas9 system. Mutation efficiency varied with genetic background in the T_0 generation, and GPC in the T_1 generation decreased significantly,owing mainly to a reduction in glutelin content. Amylose content was down-regulated significantly in some Osaap6 and all Osaap10 mutants. The increased taste value of these mutants was supported by Rapid Visco Analysis(RVA) profiles, which showed higher peak viscosity and breakdown viscosity and lower setback viscosity than the wild type. There were no significant deficiencies in agronomic traits of the mutants. Targeted mutagenesis of OsAAP6 and OsAAP10, especially OsAAP10, using the CRISPR/Cas9 system can rapidly reduce GPC and improve ECQ of rice, providing a new strategy for the breeding cultivars with desired ECQ.
文摘基因组定点编辑技术是研究基因功能和生物体改造的重要工具。CRISPR-Cas(Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins)系统是近年来发展的一种新型基因组编辑技术,该技术通过一段向导RNA和配套的核酸酶就可对特定的基因组序列进行定点编辑,具有简单高效、应用广泛的特点,受到了生物学家的广泛关注。本文着重介绍CRISPR-Cas系统在植物中的研究进展,包括CRISPR-Cas9系统在植物中的应用与完善、扩大基因组编辑范围的研究、Cas9切口酶和失活酶的拓展、特异性单碱基突变编辑系统的研究、无外源DNA污染的植物基因编辑技术的发展以及基因组编辑技术在作物育种上的应用等方面。同时也提出了还需解决的问题,并展望了基因组编辑系统在作物育种中的应用前景,为开展这一领域的研究工作提供参考。
文摘The CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPRassociated proteins) system was first identified in bacteria and archaea and can degrade exogenous substrates. It was developed as a gene editing technology in 2013. Over the subsequent years, it has received extensive attention owing to its easy manipulation, high efficiency, and wide application in gene mutation and transcriptional regulation in mammals and plants. The process of CRISPR/Cas is optimized constantly and its application has also expanded dramatically. Therefore, CRISPR/Cas is considered a revolutionary technology in plant biology. Here, we introduce the mechanism of the type II CRISPR/Cas called CRISPR/Cas9, update its recent advances in various applications in plants, and discuss its future prospects to provide an argument for its use in the study of medicinal plants.
文摘近年来,CRISPR-Cas系统病原核酸检测技术凭借其靶向精准、灵敏度高以及多重检测等特点,在即时检测(Point of Care testing,POCT)领域受到广泛关注。多种Cas蛋白具有顺式基因编辑功能和非特异性反式切割活性,基于这种特性成功实现了病原核酸检测,极大改善了POCT产品灵敏度低的问题。其中,以Cas12为靶点并反式切割单链DNA探针的系统既简单又高效。因此,就CRISPR-Cas12a的历史、分类、检测原理和在核酸检测中的应用进展、开发检测试剂的困难等方面做一综述。
