Genome editing

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    Artificial evolution of OsEPSPS through an improved dual cytosine and adenine base editor generated a novel allele conferring rice glyphosate tolerance
    Chen Zhang, Xue Zhong, Shaoya Li, Lei Yan, Jingying Li, Yubing He, Yong Lin, Yangjun Zhang and Lanqin Xia
    J Integr Plant Biol 2023, 65 (9): 2194-2203.  
    doi: 10.1111/jipb.13543
    Abstract (Browse 226)  |   Save
    Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here, through fusions of different effective cytosine and adenine deaminases with nCas9-NG, we engineered an effective surrogate two-component composite base editing system, STCBE-2, with improved C-to-T and A-to-G base editing efficiency and expanded the editing window. Furthermore, we targeted a rice endogenous OsEPSPS gene for artificial evolution through STCBE-2-mediated near-saturated mutagenesis. After hygromycin and glyphosate selection, we identified a novel OsEPSPS allele with an Asp-213-Asn (D213N) mutation (OsEPSPS-D213N) in the predicted glyphosate-binding domain, which conferred rice plants reliable glyphosate tolerance and had not been reported or applied in rice breeding. Collectively, we developed a novel dual base editor which will be valuable for artificial evolution of important genes in crops. And the novel glyphosate-tolerant rice germplasm generated in this study will benefit weeds management in rice paddy fields.
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    An efficient CRISPR/Cas9 platform for targeted genome editing in rose (Rosa hybrida)
    Chengpeng Wang, Yang Li, Na Wang, Qin Yu, Yonghong Li, Junping Gao, Xiaofeng Zhou and Nan Ma
    J Integr Plant Biol 2023, 65 (4): 895-899.  
    doi: 10.1111/jipb.13421
    Abstract (Browse 344)  |   Save
    The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐related nuclease 9 (Cas9) system enables precise, simple editing of genes in many animals and plants. However, this system has not been applied to rose (Rosa hybrida) due to the genomic complexity and lack of an efficient transformation technology for this plant. Here, we established a platform for screening single‐guide RNAs (sgRNAs) with high editing efficiency for CRISPR/Cas9‐mediated gene editing in rose using suspension cells. We used the Arabidopsis thaliana U6‐29 promoter, which showed high activity for driving sgRNA expression, to modify the CRISPR/Cas9 system. We used our highly efficient optimized CRISPR/Cas9 system to successfully edit RhEIN2, encoding an indispensable component of the ethylene signaling pathway, resulting in ethylene insensitivity in rose. Our optimized CRISPR/Cas9 system provides a powerful toolbox for functional genomics, molecular breeding, and synthetic biology in rose.
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    Cited: Web of Science(4)
      
    High-throughput genome editing in rice with a virus-based surrogate system
    Yifu Tian, Dating Zhong, Xinbo Li, Rundong Shen, Han Han, Yuqin Dai, Qi Yao, Xuening Zhang, Qi Deng, Xuesong Cao, Jian-Kang Zhu and Yuming Lu
    J Integr Plant Biol 2023, 65 (3): 646-655.  
    DOI: 10.1111/jipb.13381
    Abstract (Browse 244)  |   Save
    With the widespread use of clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) technologies in plants, large-scale genome editing is increasingly needed. Here, we developed a geminivirus-mediated surrogate system, called Wheat Dwarf Virus-Gate (WDV-surrogate), to facilitate high-throughput genome editing. WDV-Gate has two parts: one is the recipient callus from a transgenic rice line expressing Cas9 and a mutated hygromycin-resistant gene (HygM) for surrogate selection; the other is a WDV-based construct expressing two single guide RNAs (sgRNAs) targeting HygM and a gene of interest, respectively. We evaluated WDV-Gate on six rice loci by producing a total of 874 T0 plants. Compared with the conventional method, the WDV-Gate system, which was characterized by a transient and high level of sgRNA expression, significantly increased editing frequency (66.8% vs. 90.1%), plantlet regeneration efficiency (2.31-fold increase), and numbers of homozygous-edited plants (36.3% vs. 70.7%). Large-scale editing using pooled sgRNAs targeting the SLR1 gene resulted in a high editing frequency of 94.4%, further demonstrating its feasibility. We also tested WDV-Gate on sequence knock-in for protein tagging. By co-delivering a chemically modified donor DNA with the WDV-Gate plasmid, 3xFLAG peptides were successfully fused to three loci with an efficiency of up to 13%. Thus, by combining transiently expressed sgRNAs and a surrogate selection system, WDV-Gate could be useful for high-throughput gene knock-out and sequence knock-in.
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    Cited: Web of Science(2)
      
    Plant base editing and prime editing: The current status and future perspectives
    Jingying Li, Chen Zhang, Yubing He, Shaoya Li, Lei Yan, Yucai Li, Ziwei Zhu and Lanqin Xia
    J Integr Plant Biol 2023, 65 (2): 444-467.  
    doi: 10.1111/jipb.13425
    Abstract (Browse 235)  |   Save
    Precise replacement of an allele with an elite allele controlling an important agronomic trait in a predefined manner by gene editing technologies is highly desirable in crop improvement. Base editing and prime editing are two newly developed precision gene editing systems which can introduce the substitution of a single base and install the desired short indels to the target loci in the absence of double-strand breaks and donor repair templates, respectively. Since their discoveries, various strategies have been attempted to optimize both base editor (BE) and prime editor (PE) in order to improve the precise editing efficacy, specificity, and expand the targeting scopes. Here, we summarize the latest development of various BEs and PEs, as well as their applications in plants. Based on these progresses, we recommend the appropriate BEs and PEs for both basic plant research and crop improvement. Moreover, we propose the perspectives for further optimization of these two editors. We envision that both BEs and PEs will become the routine and customized precise gene editing tools for both plant biological research and crop improvement in the near future.
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    Cited: Web of Science(9)
      
    Establishment of an efficient seed fluorescence reporter‐assisted CRISPR/Cas9 gene editing in maize
    Yuanyuan Yan, Jinjie Zhu, Xiantao Qi, Beijiu Cheng, Changlin Liu and Chuanxiao Xie
    J Integr Plant Biol 2021, 63 (9): 1671-1680.  
    doi: 10.1111/jipb.13086
    Abstract (Browse 435)  |   Save
    Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR‐associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While reporter‐assisted CRISPR/Cas systems can greatly facilitate the selection of genome‐edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)‐assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En‐SFR/Cas9), embryos (Em‐SFR/Cas9), or both tissues (Em/En‐SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐ SFR/Cas9 to identify plants not harboring the genome‐editing cassette but carrying the desired mutations at target genes in single genes or in small‐scale mutant libraries, and report on the successful generation of single‐target mutants and/or mutant libraries with En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐SFR/Cas9. SFR‐assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.
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    Creation of aromatic maize by CRISPR/Cas
    Yanxiao Wang, Xiaoqin Liu, Xiuxiu Zheng, Wenxia Wang, Xunqing Yin, Haifeng Liu, Changle Ma, Xiaomu Niu, Jian‐Kang Zhu and Fei Wang
    J Integr Plant Biol 2021, 63 (9): 1664-1670.  
    doi: 10.1111/jipb.13105
    Abstract (Browse 328)  |   Save
    Aroma is an important quality parameter for breeding in rice (Oryza sativa). For example, the aromatic rice varieties basmati and jasmine rice, with a popcorn-like scent, are popular worldwide and routinely command a price premium. 2-acetyl-1-pyrroline (2AP) is a key flavor compound among over 200 volatiles identified in fragrant rice. A naturally fragrant germplasm exists in multiple plant species besides rice, which all exhibit lower activity of BETAINE ALDEHYDE DEHYDROGENASE 2 (BADH2). However, no equivalent aromatic germplasm has been described in maize (Zea mays). Here, we characterized the two maize BADH2 homologs, ZmBADH2a and ZmBADH2b. We generated zmbadh2a and zmbadh2b single mutants and the zmbadh2a-zmbadh2b double mutant by CRISPR/Cas in four inbred lines. A popcorn-like scent was only noticeable in seeds from the double mutant, but not from either single mutant or in wild type. In agreement, we only detected 2AP in fresh kernels and dried mature seeds from the double mutant, which accumulated between 0.028 and 0.723 mg/kg 2AP. These results suggest that ZmBADH2a and ZmBADH2b redundantly participate in 2AP biosynthesis in maize, and represent the creation of the world's first aromatic maize by simultaneous genome editing of the two BADH2 genes.
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    Engineering broad-spectrum disease-resistant rice by editing multiple susceptibility genes
    Hui Tao, Xuetao Shi, Feng He, Dan Wang, Ning Xiao, Hong Fang, Ruyi Wang, Fan Zhang, Min Wang, Aihong Li, Xionglun Liu, Guo‐Liang Wang and Yuese Ning
    J Integr Plant Biol 2021, 63 (9): 1639-1648.  
    doi: 10.1111/jipb.13145
    Abstract (Browse 337)  |   Save
    Rice blast and bacterial blight are important diseases of rice (Oryza sativa) caused by the fungus Magnaporthe oryzae and the bacterium Xanthomonas oryzae pv. oryzae (Xoo), respectively. Breeding rice varieties for broad-spectrum resistance is considered the most effective and sustainable approach to controlling both diseases. Although dominant resistance genes have been extensively used in rice breeding and production, generating disease-resistant varieties by altering susceptibility (S) genes that facilitate pathogen compatibility remains unexplored. Here, using CRISPR/Cas9 technology, we generated loss-of-function mutants of the S genes Pi21 and Bsr-d1 and showed that they had increased resistance to M. oryzae. We also generated a knockout mutant of the S gene Xa5 that showed increased resistance to Xoo. Remarkably, a triple mutant of all three S genes had significantly enhanced resistance to both M. oryzae and Xoo. Moreover, the triple mutant was comparable to the wild type in regard to key agronomic traits, including plant height, effective panicle number per plant, grain number per panicle, seed setting rate, and thousand-grain weight. These results demonstrate that the simultaneous editing of multiple S genes is a powerful strategy for generating new rice varieties with broad-spectrum resistance.
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    Increasing yield potential through manipulating of an ARE1 ortholog related to nitrogen use efficiency in wheat by CRISPR/Cas9
    Jiahui Zhang, Huating Zhang, Shaoya Li, Jingying Li, Lei Yan and Lanqin Xia
    J Integr Plant Biol 2021, 63 (9): 1649-1663.  
    doi: 10.1111/jipb.13151
    Abstract (Browse 321)  |   Save
    Wheat (Triticum aestivum L.) is a staple food crop consumed by more than 30% of world population. Nitrogen (N) fertilizer has been applied broadly in agriculture practice to improve wheat yield to meet the growing demands for food production. However, undue N fertilizer application and the low N use efficiency (NUE) of modern wheat varieties are aggravating environmental pollution and ecological deterioration. Under nitrogen-limiting conditions, the rice (Oryza sativa) abnormal cytokinin response1 repressor1 (are1) mutant exhibits increased NUE, delayed senescence and consequently, increased grain yield. However, the function of ARE1 ortholog in wheat remains unknown. Here, we isolated and characterized three TaARE1 homoeologs from the elite Chinese winter wheat cultivar ZhengMai 7698. We then used CRISPR/Cas9-mediated targeted mutagenesis to generate a series of transgene-free mutant lines either with partial or triple-null taare1 alleles. All transgene-free mutant lines showed enhanced tolerance to N starvation, and showed delayed senescence and increased grain yield in field conditions. In particular, the AABBdd and aabbDD mutant lines exhibited delayed senescence and significantly increased grain yield without growth defects compared to the wild-type control. Together, our results underscore the potential to manipulate ARE1 orthologs through gene editing for breeding of high-yield wheat as well as other cereal crops with improved NUE.
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    Large chromosomal segment deletions by CRISPR/LbCpf1-mediated multiplex gene editing in soybean
    Kaixuan Duan, Yuanyuan Cheng, Jing Ji, Chenchen Wang, Yongshu Wei and Yuanchao Wang
    J Integr Plant Biol 2021, 63 (9): 1620-1631.  
    doi: 10.1111/jipb.13158
    Abstract (Browse 287)  |   Save
    The creation of new soybean varieties has been limited by genomic duplication and redundancy. Efficient multiplex gene editing and large chromosomal segment deletion through clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems are promising strategies for overcoming these obstacles. CRISPR/Cpf1 is a robust tool for multiplex gene editing. However, large chromosomal excision mediated by CRISPR/Cpf1 has been reported in only a few non-plant species. Here, we report on CRISPR/LbCpf1-induced large chromosomal segment deletions in soybean using multiplex gene targeting. The CRISPR/LbCpf1 system was optimized for direct repeat and guide RNA lengths in crispr RNA (crRNA) array. The editing efficiency was evaluated using LbCpf1 driven by the CaMV35S and soybean ubiquitin promoter. The optimized system exhibited editing efficiencies of up to 91.7%. Our results showed eight gene targets could be edited simultaneously in one step when a single eight-gRNA-target crRNA array was employed, with an efficiency of up to 17.1%. We successfully employed CRISPR/LbCpf1 to produce small fragments (<1 Kb) and large chromosomal segment deletions (10 Kb–1 Mb) involving four different gene clusters in soybean. Together, these data demonstrate the power of the CRISPR/LbCpf1 platform for multiplex gene editing and chromosomal segment deletion in soybean, supporting the use of this technology in both basic research and agricultural applications.
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    Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses
    Liang Zhang, Tao Wang, Guangyang Wang, Aoyue Bi, Misganaw Wassie, Yan Xie, Liwen Cao, Huawei Xu, Jinmin Fu, Liang Chen, Yang Zhao and Tao Hu
    J Integr Plant Biol 2021, 63 (8): 1410-1415.  
    doi: 10.1111/jipb.13101
    Abstract (Browse 294)  |   Save
    Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for precise gene editing in plants. However, simultaneous gene editing of multiple homoeoalleles remains challenging, especially in self-incompatible polyploid plants. Here, we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue, using both CRISPR/Cas9 and LbCas12a (LbCpf1) systems. Loss-of-function mutants of FaPDS exhibited albino leaves, while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue. Moreover, these mutations were inheritable. Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.
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    Primary root and root hair development regulation by OsAUX4 and its participation in the phosphate starvation response
    Rigui Ye, Yunrong Wu, Zhenyu Gao, Hao Chen, Lixia Jia, Dongming Li, Xugang Li, Qian Qian and Yanhua Qi
    J Integr Plant Biol 2021, 63 (8): 1555-1567.  
    DOI: 10.1111/jipb.13142
    Abstract (Browse 275)  |   Save
    Among the five members of AUX1/LAX genes coding for auxin carriers in rice, only OsAUX1 and OsAUX3 have been reported. To understand the function of the other AUX1/LAX genes, two independent alleles of osaux4 mutants, osaux4-1 and osaux4-2, were constructed using the CRISPR/Cas9 editing system. Homozygous osaux4-1 or osaux4-2 exhibited shorter primary root (PR) and longer root hair (RH) compared to the wild-type Dongjin (WT/DJ), and lost response to indoleacetic acid (IAA) treatment. OsAUX4 is intensively expressed in roots and localized on the plasma membrane, suggesting that OsAUX4 might function in the regulation of root development. The decreased meristem cell division activity and the downregulated expression of cell cycle genes in root apices of osaux4 mutants supported the hypothesis that OsAUX4 positively regulates PR elongation. OsAUX4 is expressed in RH, and osaux4 mutants showing longer RH compared to WT/DJ implies that OsAUX4 negatively regulates RH development. Furthermore, osaux4 mutants are insensitive to Pi starvation (-Pi) and OsAUX4 effects on the -Pi response is associated with altered expression levels of Pi starvation-regulated genes, and auxin distribution/contents. This study revealed that OsAUX4 not only regulates PR and RH development but also plays a regulatory role in crosstalk between auxin and -Pi signaling.
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    The Arabidopsis R‐SNARE protein YKT61 is essential for gametophyte development
    Ting Ma, En Li, Lu‐Shen Li, Sha Li and Yan Zhang
    J Integr Plant Biol 2021, 63 (4): 676-694.  
    DOI: 10.1111/jipb.13017
    Abstract (Browse 505)  |   Save
    Gametophyte development is a pre‐requisite for plant reproduction and seed yield; therefore, studies of gametophyte development help us understand fundamental biological questions and have potential applications in agriculture. The biogenesis and dynamics of endomembrane compartments are critical for cell survival, and their regulatory mechanisms are just beginning to be revealed. Here, we report that the Arabidopsis thaliana SNARE (soluble N‐ethylmaleimide sensitive factor attachment protein receptor) protein YKT61 is essential for both male and female gametogenesis. By using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9)‐based genome editing, we demonstrated that male and female gametophytes carrying YKT61 loss‐of‐function alleles do not survive. Specifically, loss of YKT61 function resulted in the arrest of male gametophytic development at pollen mitosis I and the degeneration of female gametophytes. A three‐base‐pair deletion in YKT61 in the ykt61‐3 mutant resulted in a single‐amino acid deletion in the longin domain of YKT61; the resulting mutant protein does not interact with multiple SNAREs and showed substantially reduced membrane association, suggesting that the N‐terminal longin domain of YKT61 plays multiple roles in its function. This study demonstrates that Arabidopsis YKT61 is essential for male and female gametogenesis and sets an example for functional characterization of essential genes with the combination of Cas9‐mediated editing and expression from a Cas9‐resistant transgene.
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    Multiplex and optimization of dCas9‐TV‐mediated gene activation in plants
    Xiangyu Xiong, Jieping Liang, Zhenxiang Li, Ben‐Qiang Gong and Jian‐Feng Li
    J Integr Plant Biol 2021, 63 (4): 634-645.  
    doi: 10.1111/jipb.13023
    Abstract (Browse 430)  |   Save
    Synthetic gene activators consisting of nuclease‐dead Cas9 (dCas9) for single‐guide RNA (sgRNA)‐directed promoter binding and a transcriptional activation domain (TAD) represent new tools for gene activation from endogenous genomic locus in basic and applied plant research. However, multiplex gene coactivation by dCas9‐TADs has not been demonstrated in whole plants. There is also room to optimize the performance of these tools. Here, we report that our previously developed gene activator, dCas9‐TV, could simultaneously upregulate OsGW7 and OsER1 in rice by up to 3,738 fold, with one sgRNA targeting to each promoter. The gene coactivation could persist to at least the fourth generation. Astonishingly, the polycistronic tRNA‐sgRNA expression under the maize ubiquitin promoter, a Pol II promoter, could cause enormous activation of these genes by up to >40,000‐fold in rice. Moreover, the yeast GCN4 coiled coil‐mediated dCas9‐TV dimerization appeared to be promising for enhancing gene activation. Finally, we successfully introduced a self‐amplification loop for dCas9‐TV expression in Arabidopsis to promote the transcriptional upregulation of AtFLS2, a previously characterized dCas9‐TV‐refractory gene with considerable basal expression. Collectively, this work illustrates the robustness of dCas9‐TV in multigene coactivation and provides broadly useful strategies for boosting transcriptional activation efficacy of dCas9‐TADs in plants.
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    Roles of DEMETER in regulating DNA methylation in vegetative tissues and pathogen resistance
    Wenjie Zeng, Huan Huang, Xueqiang Lin, Chen Zhu, Ken‐ichi Kosami, Chaofeng Huang, Huiming Zhang, Cheng‐Guo Duan, Jian‐Kang Zhu and Daisuke Miki
    J Integr Plant Biol 2021, 63 (4): 691-706.  
    doi: 10.1111/jipb.13037
    Abstract (Browse 315)  |   Save
    DNA methylation is an epigenetic mark important for genome stability and gene expression. In Arabidopsis thaliana, the 5‐methylcytosine DNA glycosylase/demethylase DEMETER (DME) controls active DNA demethylation during the reproductive stage; however, the lethality of loss‐of‐function dme mutations has made it difficult to assess DME function in vegetative tissues. Here, we edited DME using clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR‐associated protein 9 and created three weak dme mutants that produced a few viable seeds. We also performed central cell‐specific complementation in a strong dme mutant and combined this line with mutations in the other three Arabidopsis demethylase genes to generate the dme ros1 dml2 dml3 (drdd) quadruple mutant. A DNA methylome analysis showed that DME is required for DNA demethylation at hundreds of genomic regions in vegetative tissues. A transcriptome analysis of the drdd mutant revealed that DME and the other three demethylases are important for plant responses to biotic and abiotic stresses in vegetative tissues. Despite the limited role of DME in regulating DNA methylation in vegetative tissues, the dme mutants showed increased susceptibility to bacterial and fungal pathogens. Our study highlights the important functions of DME in vegetative tissues and provides valuable genetic tools for future investigations of DNA demethylation in plants.
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    Pathogen‐informed breeding for crop disease resistance
    Qi Li, Bi Wang, Jinping Yu and Daolong Dou
    J Integr Plant Biol 2021, 63 (2): 305-311.  
    doi: 10.1111/jipb.13029
    Abstract (Browse 310)  |   Save
    The development of durable and broad‐spectrum resistance is an economical and eco‐friendly approach to control crop diseases for sustainable agricultural production. Emerging knowledge of the molecular basis of pathogenesis and plant–pathogen interactions has contributed to the development of novel pathogen‐informed breeding strategies beyond the limits imposed by conventional breeding. Here, we review the current status of pathogen‐assisted resistance‐related gene cloning. We also describe how pathogen effector proteins can be used to identify resistance resources and to inform cultivar deployment. Finally, we summarize the main approaches for pathogen‐directed plant improvement, including transgenesis and genome editing. Thus, we describe the emerging role of pathogen‐related studies in the breeding of disease‐resistant varieties, and propose innovative pathogen‐informed strategies for future applications.
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    Genome editing for plant research and crop improvement
    Xiangqiang Zhan, Yuming Lu, Jian-Kang Zhu and Jose Ramon Botella
    J Integr Plant Biol 2021, 63 (1): 3-33.  
    doi: 10.1111/jipb.13063
    Abstract (Browse 802)  |   Save
    The advent of clustered regularly interspaced short palindromic repeat (CRISPR) has had a profound impact on plant biology, and crop improvement. In this review, we summarize the state‐of‐the‐art development of CRISPR technologies and their applications in plants, from the initial introduction of random small indel (insertion or deletion) mutations at target genomic loci to precision editing such as base editing, prime editing and gene targeting. We describe advances in the use of class 2, types II, V, and VI systems for gene disruption as well as for precise sequence alterations, gene transcription, and epigenome control.
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    Targeted mutagenesis in Arabidopsis thaliana using CRISPR-Cas12b/C2c1
    Fan Wu , Xinyu Qiao , Yafei Zhao , Ziyi Zhang , Yifan Gao, Lingfeng Shi , Haokun Du , Lulu Wang , Ya‐Jie Zhang , Yu Zhang , Langyu Liu, Quan Wang and Dejing Kong
    J Integr Plant Biol 2020, 62 (11): 1653-1658.  
    doi: 10.1111/jipb.12944
    Abstract (Browse 532)  |   Save

    Cas12b/C2c1 is a newly identified class 2 CRISPR endonuclease that was recently engineered for targeted genome editing in mammals and rice. To explore the potential applications of the CRISPR‐Cas12b system in the dicot Arabidopsis thaliana, we selected BvCas12b and BhCas12b v4 for analysis. We successfully used both endonucleases to induce mutations, perform multiplex genome editing, and create large deletions at multiple loci. No significant mutations were detected at potential off‐target sites. Analysis of the insertion/deletion frequencies and patterns of mutants generated via targeted gene mutagenesis highlighted the potential utility of CRISPR‐Cas12b systems for genome editing in Arabidopsis.

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    A novel CCCH‐type zinc finger protein SAW1 activates OsGA20ox3 to regulate gibberellin homeostasis and anther development in rice
    Bin Wang, Ruiqiu Fang, Faming Chen, Jingluan Han, Yao-Guang Liu, Letian Chen and Qinlong Zhu
    J Integr Plant Biol 2020, 62 (10): 1594-1606.  
    DOI: 10.1111/jipb.12924
    Abstract (Browse 508)  |   Save

    Male sterility is a prerequisite for hybrid seed production. The phytohormone gibberellin (GA) is involved in regulating male reproductive development, but the mechanism underlying GA homeostasis in anther development remains less understood. Here, we report the isolation and characterization of a new positive regulator of GA homeostasis, swollen anther wall 1 (SAW1), for anther development in rice (Oryza sativa L.). Rice plants carrying the recessive mutant allele saw1 produces abnormal anthers with swollen anther wall and aborted pollen. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRIPSR‐associated protein 9‐mediated knockout of SAW1 in rice generated similar male sterile plants. SAW1 encodes a novel nucleus‐localizing CCCH‐tandem zinc finger protein, and this protein could directly bind to the promoter region of the GA synthesis gene OsGA20ox3 to induce its anther‐specific expression. In the saw1 anther, the significantly decreased OsGA20ox3 expression resulted in lower bioactive GA content, which in turn caused the lower expression of the GA‐inducible anther‐regulator gene OsGAMYB. Thus, our results disclose the mechanism of the SAW1GA20ox3‐GAMYB pathway in controlling rice anther development, and provide a new target gene for the rapid generation of male sterile lines by genome editing for hybrid breeding.

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    Expanding the scope of CRISPR/Cas9-mediated genome editing in plants using an xCas9 and Cas9-NG hybrid
    Qingfeng Niu, Siqun Wu, Yansha Li, Xiaoxuan Yang, Ping Liu, Yaping Xu and Zhaobo Lang
    J Integr Plant Biol 2020, 62 (4): 398-402.  
    doi: 10.1111/jipb.12886
    Abstract (Browse 314)  |   Save

    The widely used Streptococcus pyogenes Cas9 (SpCas9) requires NGG as a protospacer adjacent motif (PAM) for genome editing. Although SpCas9 is a powerful genome‐editing tool, its use has been limited on the targetable genomic locus lacking NGG PAM. The SpCas9 variants xCas9 and Cas9‐NG have been developed to recognize NG, GAA, and GAT PAMs in human cells. Here, we show that xCas9 cannot recognize NG PAMs in tomato, and Cas9‐NG can recognize some of our tested NG PAMs in the tomato and Arabidopsis genomes. In addition, we engineered SpCas9 (XNG‐Cas9) based on mutations from both xCas9 and Cas9‐NG, and found that XNG‐Cas9 can efficiently mutagenize endogenous target sites with NG, GAG, GAA, and GAT PAMs in the tomato or Arabidopsis genomes. The PAM compatibility of XNG‐Cas9 is the broadest reported to date among Cas9s (SpCas9 and Cas9‐NG) active in plant.

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    Application and future perspective of CRISPR/Cas9 genome editing in fruit crops
    Junhui Zhou, Dongdong Li, Guoming Wang, Fuxi Wang, Merixia Kunjal, Dirk Joldersma and Zhongchi Liu
    J Integr Plant Biol 2020, 62 (3): 269-286.  
    doi: 10.1111/jipb.12793
    Abstract (Browse 438)  |   Save

    Fruit crops, including apple, orange, grape, banana, strawberry, watermelon, kiwifruit and tomato, not only provide essential nutrients for human life but also contribute to the major agricultural output and economic growth of many countries and regions in the world. Recent advancements in genome editing provides an unprecedented opportunity for the genetic improvement of these agronomically important fruit crops. Here, we summarize recent reports of applying CRISPR/Cas9 to fruit crops, including efforts to reduce disease susceptibility, change plant architecture or flower morphology, improve fruit quality traits, and increase fruit yield. We discuss challenges facing fruit crops as well as new improvements and platforms that could be used to facilitate genome editing in fruit crops, including dCas9‐base‐editing to introduce desirable alleles and heat treatment to increase editing efficiency. In addition, we highlight what we see as potentially revolutionary development ranging from transgene‐free genome editing to de novo domestication of wild relatives. Without doubt, we now see only the beginning of what will eventually be possible with the use of the CRISPR/Cas9 toolkit. Efforts to communicate with the public and an emphasis on the manipulation of consumer‐friendly traits will be critical to facilitate public acceptance of genetically engineered fruits with this new technology.

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    Developing disease-resistant thermosensitive male sterile rice by multiplex gene editing
    Sanfeng Li, Lan Shen, Ping Hu, Qing Liu, Xudong Zhu, Qian Qian, Kejian Wang and Yuexing Wang
    J Integr Plant Biol 2019, 61 (12): 1201-1205.  
    doi: 10.1111/jipb.12774
    Abstract (Browse 1576)  |   Save
    High‐quality and disease‐resistant male sterile lines have great potential for applications in hybrid rice breeding. We introduced specific mutations into the TMS5, Pi21, and Xa13 genes in Pinzhan intermediate breeding material using the CRISPR/Cas9 multiplex genome editing system. We found that the transgene‐free homozygous triple tms5/pi21/xa13 mutants obtained in the T1 generation displayed characteristics of thermosensitive genic male sterility (TGMS) with enhanced resistance to rice blast and bacterial blight. Our study provides a convenient and effective way of converting breeding intermediate material into TGMS lines through multiplex gene editing, which could significantly accelerate the breeding of sterile lines.
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