Improving the Genome Editing Efficiency of CRISPR/Cas9 in Melon and Watermelon DOI Creative Commons
Zhuanrong Wang, Lili Wan, Jian Ren

et al.

Cells, Journal Year: 2024, Volume and Issue: 13(21), P. 1782 - 1782

Published: Oct. 28, 2024

CRISPR/Cas9 is a powerful genome editing tool for trait improvement in various crops; however, enhancing mutation efficiency using watermelon and melon remains challenging. We designed four CRISPR systems with different sgRNA expression cassettes to target the phytoene desaturase (PDS) gene melon. The constructed vectors were delivered host plants Agrobacterium-mediated transformation. Phenotypic genotypic analyses of edited seedlings revealed that tRNA Csy4 spacers driven by Pol II-type promoter significantly improved efficiency, reaching 25.20% 42.82%, respectively. Notably, 78.95% mutations generated system involved large-fragment deletions (LDs) between two sites. In watermelon, achieved PDS 41.48%, 71.43% showing LD Sequencing analysis indicated exhibited heterozygous, three-allele chimeric events; included 2/14 homozygous mutations. Compared commonly used III promoter, II drive containing showed best melon; this was also effective watermelon.

Language: Английский

Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein in the T0 generation DOI Creative Commons
Hang Su, Yuanchun Wang, Jin Xu

et al.

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: July 4, 2023

Citrus canker caused by Xanthomonas citri subsp. (Xcc) is a destructive citrus disease worldwide. Generating disease-resistant cultivars the most effective, environmentally friendly and economic approach for control. However, traditional breeding lengthy laborious. Here, we develop transgene-free canker-resistant sinensis lines in T0 generation within 10 months through transformation of embryogenic protoplasts with Cas12a/crRNA ribonucleoprotein to edit susceptibility gene CsLOB1. Among 39 regenerated lines, 38 are biallelic/homozygous mutants, demonstrating 97.4% mutation rate. No off-target mutations detected edited lines. Canker resistance cslob1-edited results from both abolishing symptoms inhibiting Xcc growth. The C. have received regulatory approval USDA APHIS exempted EPA regulation. This study provides sustainable efficient control solution presents an genome-editing strategy other crops.

Language: Английский

Citations

43
Sweet Orange: Evolution, Characterization, Varieties, and Breeding Perspectives DOI Creative Commons
S. Seminara, Stefania Bennici, Mario Di Guardo

et al.

Agriculture, Journal Year: 2023, Volume and Issue: 13(2), P. 264 - 264

Published: Jan. 21, 2023

Among Citrus species, the sweet orange (Citrus sinensis (L.) Osbeck) is most important in terms of production volumes and cultivated areas. Oranges are particularly appreciated for organoleptic characteristics high nutraceutical value fruits (thanks especially to their content antioxidants). Recent advances citrus genetic genomic resources, such as release reference genomes several cultivars, have contributed (i) understanding diversification C. its relation with other (ii) assessing molecular mechanisms underlying traits interest, (iii) identifying characterizing candidate genes responsible phenotypic traits, (iv) developing biotechnological methods incorporate these into different genotypes. It has been clarified that all diversity within species was derived from subsequent mutations starting a single ancestor complex cycles hybridization backcrossing between mandarin reticulata Blanco) pummelo maxima (Burm.) Merr.). This paper provides an overview varietal panorama together description main driving forces present future breeding. In fact, orange, well novel varieties improved being pursued thanks employment conventional and/or innovative (molecular-based) methods. The state art innovations genomics tools leading so-called new plant breeding technologies were also reviewed discussed.

Language: Английский

Citations

36
Transgene-free genome editing of vegetatively propagated and perennial plant species in the T0 generation via a co-editing strategy DOI
Xiaoen Huang,

Hongge Jia,

Jin Xu

et al.

Nature Plants, Journal Year: 2023, Volume and Issue: 9(10), P. 1591 - 1597

Published: Sept. 18, 2023

Language: Английский

Citations

31
CRISPR technology towards genome editing of the perennial and semi-perennial crops citrus, coffee and sugarcane DOI Creative Commons
Guilherme Souza Prado,

Dhiôvanna Corrêia Rocha,

Lucas Nascimento dos Santos

et al.

Frontiers in Plant Science, Journal Year: 2024, Volume and Issue: 14

Published: Jan. 8, 2024

Gene editing technologies have opened up the possibility of manipulating genome any organism in a predicted way. CRISPR technology is most used tool and, agriculture, it has allowed expansion possibilities plant biotechnology, such as gene knockout or knock-in, transcriptional regulation, epigenetic modification, base editing, RNA prime and nucleic acid probing detection. This mostly depends on

Language: Английский

Citations

14
CRISPR/Cas9-generated mutations in a sugar transporter gene reduce cassava susceptibility to bacterial blight DOI Creative Commons
Kiona Elliott, Kira M. Veley, Greg Jensen

et al.

PLANT PHYSIOLOGY, Journal Year: 2024, Volume and Issue: 195(4), P. 2566 - 2578

Published: May 3, 2024

Bacteria from the genus Xanthomonas are prolific phytopathogens that elicit disease in over 400 plant species. Xanthomonads carry a repertoire of specialized proteins called transcription activator-like (TAL) effectors promote and pathogen virulence by inducing expression host susceptibility (S) genes. phaseoli pv. manihotis (Xpm) causes bacterial blight on staple food crop cassava (Manihot esculenta Crantz). The Xpm effector TAL20 induces ectopic S gene Manihot Sugars Will Eventually be Exported Transporter 10a (MeSWEET10a), which encodes sugar transporter contributes to (CBB) susceptibility. We used CRISPR/Cas9 generate multiple lines with edits MeSWEET10a binding site and/or coding sequence. In several regenerated lines, was no longer induced Xpm, these cases, we observed reduced CBB symptoms post infection. Because is expressed flowers, further characterized reproductive capability promoter sequence mutants. Lines were crossed themselves wild-type plants. results indicated female, but not male, flowers critical produce viable F1 seed. case mutations left intact, progeny recovered. Taken together, demonstrate blocking induction strategy for decreasing ideal will contain block TAL while leaving endogenous unaltered.

Language: Английский

Citations

9
Targeted MYC2 stabilization confers citrus Huanglongbing resistance DOI
Pingzhi Zhao, Huan Yang, Yanwei Sun

et al.

Science, Journal Year: 2025, Volume and Issue: 388(6743), P. 191 - 198

Published: April 10, 2025

Huanglongbing (HLB) is a devastating citrus disease. In this work, we report an HLB resistance regulatory circuit in Citrus composed of E3 ubiquitin ligase, PUB21, and its substrate, the MYC2 transcription factor, which regulates jasmonate-mediated defense responses. A helitron insertion PUB21 promoter introduced multiple MYC2-binding cis-elements to create linking activity with degradation. Ectopic expression natural dominant-negative paralog discovered distant relatives stabilized conferred HLB. Antiproteolysis peptides (APPs), identified by artificial intelligence, binding inhibiting activity. 14–amino acid peptide, APP3-14, molecularly controlled greenhouse field trials. This approach represents strategy combat uncultivable pathogens through targeted disease protein stabilization.

Language: Английский

Citations

1
Base Editors for Citrus Gene Editing DOI Creative Commons
Xiaoen Huang, Yuanchun Wang, Nian Wang

et al.

Frontiers in Genome Editing, Journal Year: 2022, Volume and Issue: 4

Published: Feb. 28, 2022

Base editors, such as adenine base editors (ABE) and cytosine (CBE), provide alternatives for precise genome editing without generating double-strand breaks (DSBs), thus avoiding the risk of instability unpredictable outcomes caused by DNA repair. Precise gene mediated in citrus has not been reported. Here, we have successfully adapted ABE to edit TATA box promoter region canker susceptibility

Language: Английский

Citations

31
Multi-faceted CRISPR-Cas9 strategy to reduce plant based food loss and waste for sustainable bio-economy – A review DOI
P. Hemalatha, Ebrahim M. Abda, Shipra Shah

et al.

Journal of Environmental Management, Journal Year: 2023, Volume and Issue: 332, P. 117382 - 117382

Published: Feb. 6, 2023

Language: Английский

Citations

22
Generation of transgene-free canker-resistant Citrus sinensis cv. Hamlin in the T0 generation through Cas12a/CBE co-editing DOI Creative Commons

Hongge Jia,

Ahmad A. Omar, Jin Xu

et al.

Frontiers in Plant Science, Journal Year: 2024, Volume and Issue: 15

Published: March 26, 2024

Citrus canker disease affects citrus production. This is caused by Xanthomonas citri subsp. (Xcc). Previous studies confirmed that during Xcc infection, PthA4, a transcriptional activator like effector (TALE), translocated from the pathogen to host plant cells. PthA4 binds binding elements (EBEs) in promoter region of susceptibility gene LOB1 (EBE -LOBP) activate its expression and subsequently cause symptoms. Previously, Cas12a/CBE co-editing method was employed disrupt EBE -LOBP pummelo, which highly homozygous. However, most commercial cultivars are heterozygous hybrids more difficult generate homozygous/biallelic mutants. Here, we edit Hamlin ( sinensis ), hybrid cultivar grown worldwide. Binary vector GFP-p1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1 constructed shown be functional via Xcc-facilitated agroinfiltration leaves. construct allows selection transgene-free regenerants GFP, edits ALS chlorsulfuron-resistant as marker for genome editing resulting transient T-DNA nCas9-mPBE:ALS2:ALS1, gene(s) interest (i.e., this study) through ttLbCas12a, thus creating citrus. Totally, 77 plantlets were produced. Among them, 8 transgenic plants (#Ham GFP 1 - #Ham 8), 4 NoGFP 4), rest wild type. plantlets, three lines 1, 2 3) contained biallelic mutations pthA4 , one line 4) had homozygous . We achieved 5.2% mutation efficiency –LOBP C. cv. Hamlin, compared 1.9% pummelo previous study. Importantly, four 3 survived resistant against canker. Taken together, has been successfully used canker‐resistant T0 generation biallelic/homozygous LOB1.

Language: Английский

Citations

7
LbCas12a-D156R Efficiently Edits LOB1 Effector Binding Elements to Generate Canker-Resistant Citrus Plants DOI Creative Commons

Hongge Jia,

Yuanchun Wang, Hang Su

et al.

Cells, Journal Year: 2022, Volume and Issue: 11(3), P. 315 - 315

Published: Jan. 18, 2022

Citrus canker caused by Xanthomonas citri subsp. (Xcc) is an economically important disease in most citrus production regions worldwide. Xcc secretes a transcriptional activator like effector (TALE) PthA4 to bind the binding elements (EBEs) promoter region of susceptibility gene LOB1 activate its expression, which turn causes symptoms. Editing EBE with Cas9/gRNA has been used generate resistant plants. However, EBE-edited lines generated contain indels 1-2 bp, higher possibility be overcome adaptation. The adaptation capacity TALEs inversely correlates number mismatches EBE. LbCas12a/crRNA known longer deletion than Cas9. In this study, we temperature-tolerant and more efficient LbCas12a variant (ttLbCas12a), harboring single substitution D156R, modify LOB1. We first constructed GFP-p1380N-ttLbCas12a:LOBP, was shown functional via Xcc-facilitated agroinfiltration Pummelo (Citrus maxima) leaves. Subsequently, stably expressed ttLbCas12a:LOBP Pummelo. Eight transgenic were generated, seven showing 100% mutations EBE, among one line homozygous. had ttLbCas12a-mediated deletions up 10 bp. Importantly, no off-targets detected. summary, ttLbCas12a can efficiently biallelic/homozygous mutant short deletions, thus providing useful tool for study breeding citrus.

Language: Английский

Citations

26