Increasing the efficiency of CRISPR‐Cas9‐VQR precise genome editing in rice

Hu, Xixun; Meng, Xiangbing; Liu, Qing; Li, Jiayang; Wang, Kejian

doi:10.1111/pbi.12771

Cited by 74 publications

(51 citation statements)

References 33 publications

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“…The plasmids expressing the CRISPR/Cas9 system were constructed via the isocaudamer ligation method, as previously described 11 . The modified single guide RNAs (sgRNAs) scaffold and ACTIN1 promoter-driven Cas9 were used to increase the mutation rate in this study 12 . Briefly, the double-stranded overhangs of target oligoes (listed in Extended Data Table1) were ligated into the SK-sgRNA vectors digested with Aar I.…”

Section: Methodsmentioning

confidence: 99%

Clonal seeds in hybrid rice using CRISPR/Cas9

Shen

et al. 2018

Preprint

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12 13 2 Heterosis, the observation that first generation hybrids outcompete the parental 14 lines, is widely used in increasing the productivity and yield of agricultural 15 crops 1,2 . However, heterosis is lost in the following generations because of genetic 16 segregation. In addition, the high cost of hybrid seed production hinders the 17 application of heterosis in many crops. Clonal reproduction through seeds could 18 be revolutionary for agriculture by allowing self-propagation of F1 hybrids 3,4 . 19Here we show that heterozygosity of F1 hybrid rice can be fixed and thus 20 propagated without additional crossing. First, we showed that multiplex editing 21 of three key meiotic genes 5,6 in hybrid rice leads to the production of clonal 22 diploid gametes and tetraploid seeds. Next, editing of the MATRILINEAL (MTL) 23 gene that involved in fertilization 7,8 results in the induction of haploid seeds in 24 hybrid rice. By simultaneous editing of these four endogenous genes in hybrid 25 rice using the CRISPR/Cas9 system, we obtained in one generation plants able to 26 propagate clonally through seeds. This opens the possibility to fix heterozygosity 27 of hybrid varieties in food crops. 28 Heterosis (also known as hybrid vigor) is a phenomenon whereby hybrid 29 offspring of genetically diverse individuals display increased vigor relative to their 30 homozygous parents. Heterosis has been widely applied in agriculture to dramatically 31improve the production and to broaden adaptability of crops 1,2 . However, the essential 32 process of hybrid seed production increases the seed cost and even prohibits its 33 application in many crops. It has been proposed to fix the heterosis of hybrid crop by 34 introduction of apomixis 3 . Apomixis is an asexual reproductive strategy where the 35 3 offspring were generated through seeds, but without meiosis and fertilization. 36 Although it has been described in many flowering plant taxa 9 , apomixis has not been 37 reported in major crops. Previously, it was revealed that combined mutations of three 38 genes that affect key meiotic processes created a genotype called MiMe (Mitosis 39 instead of Meiosis) in which meiosis is totally replaced by mitotic-like division, 40 leading to the production of male and female clonal diploid gametes in Arabidopsis 41 and rice 5,6 . However, the self-fertilization of MiMe resulted in doubling of ploidy at 42 each generation. By crossing Arabidopsis MiMe with CenH3-mediated chromosome 43 elimination line, clonal diploid offspring were obtained 4 . However, the system still 44 relies on the crossing between different plants and the CENH3-mediated chromosome 45 elimination appeared to be difficult to transfer to other species 10 . Therefore, further 46 work is required to achieve the aim of heterosis fixation in self-fertilized hybrids.47

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Section: Methodsmentioning

confidence: 99%

Clonal seeds in hybrid rice using CRISPR/Cas9

Shen

et al. 2018

Preprint

Self Cite

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“…Several reports have indicated that constitutively expressed Cas9 produces an excess of sgRNA-Cas9, which may increase the incidence of genome-wide offtarget mutations (Hsu et al 2013;Hu et al 2018;Pattanayak et al 2013;Svitashev et al 2016). By contrast, conditionally or transiently expressing Cas9 significantly reduces the frequency of off-target mutations (Srivastava et al 2017;Zhang et al 2014).…”

Section: Reducing Off-target Mutations and Lethality In Monocotsmentioning

confidence: 99%

“…The CRISPR/Cas9 system has been successfully used for genome editing in a variety of monocots, including rice (Oryza sativa), wheat (Triticum sp. ), barley (Hordeum vulgare), and maize (Zea mays) (Feng et al 2018;Gasparis et al 2018;Hu et al 2018;Kis et al 2019;Okamoto et al 2017;Wang et al 2017;Zhang et al 2016). To perform CRISPR/Cas9-mediated genome editing, the Cas9 endonuclease is guided by a single guide RNA (sgRNA) to recognize the complementary sequence and create double-strand breaks (DSBs), thereby generating a short deletion or insertion.…”

mentioning

confidence: 99%

How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis

Yue

Hong

Wei

et al. 2020

Rice

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The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; however, several factors need to be considered for the efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes, many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA ribonucleoproteins (RNPs) is one strategy that can be used to prevent transgene issues with the expression of sgRNA and Cas proteins. RNPs can be used to efficiently generate transgene-free genome-edited crops that can reduce transgene issues related to the generation of genetically modified organisms. In this review, we introduce new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. Four topics are covered: the design and construction of plasmids for genome editing in monocots; alternatives to SpCas9; protoplasts and CRISPR; and screening for marker-free CRISPR/Cas9-induced mutants. We have aimed to encompass a full spectrum of information for genome editing in monocot crops.

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“…VQR, EQR and VRER recognize 5 0 -NGAN-3 0 , 5 0 -NGAG-3 0 and 5 0 -NGCG-3 0 PAMs, respectively. In rice, the SpCas9 variants VQR and VRER have been demonstrated to recognize 5 0 -NGA-3 0 and 5 0 -NGCG-3 PAM sequences, respectively (Hu et al 2016(Hu et al , 2018a.…”

Section: Targeting Altered Pams With Cas9 Orthologs and Engineered Vamentioning

confidence: 99%

CRISPR-Cas nucleases and base editors for plant genome editing

et al. 2019

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Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) and base editors are fundamental tools in plant genome editing. Cas9 from Streptococcus pyogenes (SpCas9), recognizing an NGG protospacer adjacent motif (PAM), is a widely used nuclease for genome editing in living cells. Cas12a nucleases, targeting T-rich PAMs, have also been recently demonstrated in several plant species. Furthermore, multiple Cas9 and Cas12a engineered variants and orthologs, with different PAM recognition sites, editing efficiencies and fidelity, have been explored in plants. These RNA-guided sequence-specific nucleases (SSN) generate double-stranded breaks (DSBs) in DNA, which trigger non-homologous end-joining (NHEJ) repair or homology-directed repair (HDR), resulting in insertion and deletion (indel) mutations or precise gene replacement, respectively. Alternatively, genome editing can be achieved by base editors without introducing DSBs. So far, several base editors have been applied in plants to introduce C-to-T or A-to-G transitions, but they are still undergoing improvement in editing window size, targeting scope, off-target effects in DNA and RNA, product purity and overall activity. Here, we summarize recent progress on the application of Cas nucleases, engineered Cas variants and base editors in plants.

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Increasing the efficiency of CRISPR‐Cas9‐VQR precise genome editing in rice

Cited by 74 publications

References 33 publications

Clonal seeds in hybrid rice using CRISPR/Cas9

Clonal seeds in hybrid rice using CRISPR/Cas9

How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis

CRISPR-Cas nucleases and base editors for plant genome editing

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