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    Review
    Development and Application of CRISPR/Cas System in Rice
    Jun Ren, Xixun Hu, Kejian Wang, Chun Wang
    2019, 26(2): 69-761.  DOI: 10.1016/j.rsci.2019.01.001
    Abstract ( )   HTML ( )   PDF (1551KB) ( )  

    In the past several years, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) system has been harnessed as an efficient and powerful tool for targeted genome editing in different prokaryotic and eukaryotic species. Here, we review the development and application of CRISPR/Cas system in rice, emphasizing different varieties of CRISPR/Cas systems have been applied, strategies for multiplex editing, methods for precise gene insertion and replacement, and efficient systems for base editing and site-specific transcriptional regulation. In addition, the biosecurity of CRISPR/Cas system is also discussed, including transgene-free methods and off-target effects of CRISPR/Cas system. Thus, the development and application of CRISPR/Cas system will have significant impact on functional genomic research and variety improvement in rice.

    Research Papers
    Editing of Rice Isoamylase Gene ISA1 Provides Insights into Its Function in Starch Formation
    Shufen Chao, Yicong Cai, Baobing Feng, Guiai Jiao, Zhonghua Sheng, Ju Luo, Shaoqing Tang, Jianlong Wang, Peisong Hu, Xiangjin Wei
    2019, 26(2): 77-87.  DOI: 10.1016/j.rsci.2018.07.001
    Abstract ( )   HTML ( )   PDF (1862KB) ( )  

    Isoamylase 1 (ISA1) is an isoamylase-type debranching enzyme which plays a predominant role in amylopectin synthesis. In this study, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease 9 (CRISPR/Cas9) system was used to edit ISA1 gene in rice via Agrobacterium-mediated transformation. We identified 36 genetic edited lines from 55 T0 transgenic events, and classified the mutation forms into 7 types. Of those, two homozygous mutants, cr-isa1-1 (type 1, with an adenine insertion) and cr-isa1-2 (type 3, with a cytosine deletion) were selected for further analysis. Seed sizes of both cr-isa1-1 and cr-isa1-2 were affected, and the two mutants also displayed a shrunken endosperm with significantly lower grain weight. Electron microscopy analysis showed that abnormal starch granules and amyloplasts were found in cr-isa1-1 and cr-isa1-2 endosperm cells. The contents of total starch, amylose and amylopectin in the endosperm of the cr-isa1 mutants were significantly reduced, whereas sugar content and starch gel consistency were observably increased compared to the wild-type. The gelatinization temperature and starch chain length distributions of the cr-isa1 mutants were also altered. Moreover, transcript levels of most starch synthesis-related genes were significantly lower in cr-isa1 mutants. In conclusion, the results indicated that gene edition of ISA1 affected starch synthesis and endosperm development, and brought potential implications for rice quality breeding.

    Characterization and Evaluation of OsLCT1 and OsNramp5 Mutants Generated Through CRISPR/Cas9-Mediated Mutagenesis for Breeding Low Cd Rice
    Songmei Liu, Jie Jiang, Yang Liu, Jun Meng, Shouling Xu, Yuanyuan Tan, Youfa Li, Qingyao Shu, Jianzhong Huang
    2019, 26(2): 88-97.  DOI: 10.1016/j.rsci.2019.01.002
    Abstract ( )   HTML ( )   PDF (1776KB) ( )  

    To explore how rice (Oryza sativa L.) can be safely produced in Cd-polluted soil, OsLCT1 and OsNramp5 mutant lines were generated by CRISPR/Cas9-mediated mutagenesis. One of OsLCT1 mutant (lct1×1) and two of OsNramp5 mutants (nramp5×7 and nramp5×9) were evaluated for grain Cd accumulation and agronomic performances. In paddy field soil containing approximately 0.9 mg/kg Cd, lct1×1 grains contained approximately 40% (0.17 mg/kg) of the Cd concentration of the wild type parental line, less than the China National Food Safety Standard (0.20 mg/kg). Both OsNramp5 mutants showed low grain Cd accumulation (< 0.06 mg/kg) in the paddy (approximately 0.9 mg/kg Cd) or in pots in soil spiked with 2 mg/kg Cd. However, only nramp5×7 showed normal growth and yield, whereas the growth of nramp5×9 was severely impaired. The study showed that lct1×1 could be used to produce rice grains safe for human consumption in lightly contaminated paddy soils and nramp5×7 used in soils contaminated by much higher levels of Cd.

    Targeted Mutagenesis of NAC Transcription Factor Gene, OsNAC041, Leading to Salt Sensitivity in Rice
    Bo Wang, Zhaohui Zhong, Huanhuan Zhang, Xia Wang, Binglin Liu, Lijia Yang, Xiangyan Han, Deshui Yu, Xuelian Zheng, Chunguo Wang, Wenqin Song, Chengbin Chen, Yong Zhang
    2019, 26(2): 98-108.  DOI: 10.1016/j.rsci.2018.12.005
    Abstract ( )   HTML ( )   PDF (2723KB) ( )  

    Salinity is a major abiotic stress factor that seriously affects plant growth. Many genes are involved in the response to salt stress with various metabolism pathways. A number of plant transcription factor family genes have been found to be involved in the salt stress response, and NAM, ATAF and CUC (NAC) transcription factors are thought to act as active regulators during abiotic stress, especially salt stress. In this study, we detected a rice NAC transcription factor coding gene, OsNAC041, and confirmed that it influenced the germination of seeds under salt stress and salt tolerance of plants. OsNAC041 was primarily expressed in the leaves and located in the nucleus. Furthermore, the CRISPR/Cas9 method was used to obtain a targeted osnac041 mutant, of which the plant height was higher than that of the wild-type, showing increased salt sensitivity. Moreover, RNA-seq analysis revealed a number of differentially expressed genes (DEGs) involved in several important signaling pathways in the osnac041 mutant. Subsequently, Kyoto Encyclopedia of Genes and Genomes annotation also revealed differential expression of DEGs associated with mitogen-activated protein kinase signaling, peroxisome, eukaryotic- type ABC transporters, photosynthesis and plant hormones, which are involved in stress-related signaling pathways. Overall, our study suggested that OsNAC041 was involved in the salt stress response in rice. These findings not only provide empirical evidence of OsNAC041 function, but also provide new insight into its potential application in rice resistance breeding.

    Short Communications
    Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants
    Yubing He, Min Zhu, Lihao Wang, Junhua Wu, Qiaoyan Wang, Rongchen Wang, Yunde Zhao
    2019, 26(2): 109-117.  DOI: 10.1016/j.rsci.2018.11.001
    Abstract ( )   HTML ( )   PDF (26012KB) ( )  

    Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR (TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.

    Production of Two Elite Glutinous Rice Varieties by Editing Wx Gene
    Yunyan Fei, Jie Yang, Fangquan Wang, Fangjun Fan, Wenqi Li, Jun Wang, Yang Xu, Jinyan Zhu, Weigong Zhong
    2019, 26(2): 118-124.  DOI: 10.1016/j.rsci.2018.04.007
    Abstract ( )   HTML ( )   PDF (1474KB) ( )  

    The waxy gene (Wx) in rice, which encodes the granule bound starch synthase enzyme, is responsible for amylose synthesis. Glutinous (sticky) rice has little or no amylose that can be used in various applications, such as brewing. In this study, knockout of the Wx gene with CRISPR/Cas9 technology was conducted in two elite japonica rice lines, Huaidao 5 (HD5) and Suken 118 (SK118), aiming to develop elite sticky rice varieties. We achieved six homozygous T0 plants with more than 200 bp deletion in the Wx gene, as well as 36 wx-HD5 and 18 wx-SK118 homozygous transgene-free plants in the T1 generation. The seeds of all the mutants were white and opaque, similar to those of sticky rice, and contained only 2.6%-3.2% amylose. Results of scanning electron microscopy showed that the quality of rice did not change. In conclusion, we successfully developed two elite sticky rice varieties.

    Letters
    CRISPR/Cas9-Mediated Adenine Base Editing in Rice Genome
    Hao Li, Ruiying Qin, Xiaoshuang Liu, Shengxiang Liao, Rongfang Xu, Jianbo Yang, Pengcheng Wei
    2019, 26(2): 125-128.  DOI: 10.1016/j.rsci.2018.07.002
    Abstract ( )   HTML ( )   PDF (990KB) ( )  

    SUMMARY Precise base editing is highly desired in plant functional genomic research and crop molecular breeding. In this study, we constructed a rice-codon optimized adenine base editor (ABE)-nCas9 tool that induced targeted A?T to G?C point mutation of a key single nucleotide polymorphism site in an important agricultural gene. Combined with the modified single-guide RNA variant, our plant ABE tool can efficiently achieve adenine base editing in the rice genome.

    Rapid Creation of New Photoperiod-/Thermo-Sensitive Genic Male-Sterile Rice Materials by CRISPR/Cas9 System
    Lan Shen, Guojun Dong, Yu Zhang, Guocheng Hu, Qiang Zhang, Guanglian Hu, Bo Xu, Deyong Ren, Jiang Hu, Li Zhu, Zhenyu Gao, Guangheng Zhang, Longbiao Guo, Dali Zeng, Qian Qian
    2019, 26(2): 129-132.  DOI: 10.1016/j.rsci.2018.12.006
    Abstract ( )   HTML ( )   PDF (1238KB) ( )  

    SUMMARY Rice is an important food crop in China and the development of hybrid rice is a crucial way to increase grain yield. The creation of dual-purpose nuclear-sterile lines for two-line hybrid breeding has become vital for commercial rice breeding. We constructed the pC1300-2x35S::Cas9-sgRNAPTGMS2-1 expression vector for editing the male fertility gene PTGMS2-1 in two widely compatible rice varieties 93-11 and Huazhan by using the CRISPR/Cas9 system. We obtained the marker-free photoperiod-/thermo- sensitive genic male-sterile (P/TGMS) lines in T1 generation. According to the experiments in phytotron with four temperature and photoperiod treatments we found the temperature is the main factor for restoring the pollen fertility of ptgms2-1 mutants in 93-11 and Huazhan and the photoperiod also has some effects on pollen fertility in two different rice backgrounds. The application of cultivating new male-sterile lines by genome editing system will significantly accelerate the rice breeding process.