Augmentation of betacyanin and quercetin in hybrid callus: Comprehensive assessment of biosynthesized silver nanoparticles for their potent biological activities, advanced in silico interactions, and rigorous toxicological evaluation

Industrial Crops and Products, Journal Year: 2025, Volume and Issue: 227, P. 120824 - 120824

Published: March 14, 2025

Language: Английский

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Cleavable donor‐assisted CRISPR/Cas9 system significantly improves the efficiency of large DNA insertion in Physcomitrium patens

The Plant Journal, Journal Year: 2025, Volume and Issue: 121(4)

Published: Feb. 1, 2025

SUMMARY Precise insertion of desired fragments can be achieved by CRISPR/Cas9‐based genome editing. However, a decrease in knock‐in efficiency has been observed with increasing length exogenous inserts. In this study, we developed an vivo cleavable (IVC) donor‐assisted CRISPR/Cas9 system to improve efficiency, particularly for larger inserts, the moss Physcomitrium patens ( P. ). The IVC donor, which contains two Cas9 nuclease recognition sites flanking homology template, enables release linear template homology‐directed repair (HDR) when co‐delivered corresponding plasmid into protoplasts. our experimental framework, distinct sgRNAs and four different DNA inserts were evaluated. Compared standard circular donors, donors significantly enhanced CRISPR/Cas9‐mediated precise 5.8, 7.5, 11.1 kb at PpPDV2‐4 sgRNA target site, improving integration rates from 29.6 67.8%, 15.0 72.0%, 12.1 65.6%, respectively. At alternative sgRNA2 site within Pp6c18_3160 locus, donor also demonstrated higher 7.4 fragment compared donor. This approach large represents powerful tool basic research synthetic biology efforts species. Moreover, strategy may potentially applicable crops that are amenable protoplast transformation regeneration, facilitating improvement key traits.

Language: Английский

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Genome Editing in Legumes: Current Status

Published: Jan. 1, 2025

Language: Английский

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Optimizing Genome Editing in Mollusks (Crassostrea gigas) in Vitro Validation of sgRNA and Identifying Key Factors Influencing Efficiency

The CRISPR Journal, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 28, 2025

CRISPR-Cas9 genome editing holds tremendous potential for accelerating genetic improvements in aquaculture. The success of the system relies on specificity and efficiency engineered single-guide RNAs (sgRNAs). In this study, we optimized an vitro validation protocol sgRNAs to streamline gene process, capitalizing limited breeding season Pacific oyster (Crassostrea gigas). We evaluated 11 targeting four genes both vivo C. gigas. addition, found that Cas9 protein differs from mRNA at various stages early development. proved particular efficacy achieving efficient knockout, functioning effectively during first cell division facilitating biallelic knockouts. Statistical analysis showed group, frequency ranged 12.5% 57.8%, overall reached as high 75–90.6%. group exhibited a 3.1–14.0% spanning 65.6–78.1%. Contrary expectations, low-temperature incubation (20°C) embryos prolonged time window but did not improve efficiency, likely due temperature sensitivity enzyme activity. Together, study provides comprehensive factors affecting gigas, providing robust framework future endeavors mollusks other marine invertebrates.

Language: Английский

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Long-read genome sequencing reveals the sequence characteristics of pear self-incompatibility locus

Molecular Horticulture, Journal Year: 2025, Volume and Issue: 5(1)

Published: March 1, 2025

Abstract The S-RNase-based self-incompatibility locus ( S -locus) in Petunia species contains 16–20 F-box genes, which collaboratively function the recognition and subsequent degradation of non-self S-RNases, while distinguishing them from self S-RNase. However, number -locus F - box genes SFBB s) physically interacted with S-RNases remains uncertain Pyrus species. Utilizing Pacbio long-read sequencing, we successfully assembled genome pear cultivar ‘Yali’ bretschneideri ), identified 19 s 17 spanning approximately 1.78 Mb. Additionally, 17–21 other Malus -loci a range 1.35 to 2.64 Based on phylogenetic analysis, it was determined that could be classified into 22 groups, denoted as I XXII. At amino acid level, within given group exhibited average identities ranged 88.9% 97.9%. Notably, all co-segregated -RNase , 18 being specifically expressed pollen. Consequently, these pollen-specifically are considered potential candidates for pollen- determinant. Intriguingly, out s, eight demonstrated interactions at least one S-RNase, remaining SFBBs failed recognize any These findings provide compelling evidence supporting existence collaborative non-self-recognition system governing

Language: Английский

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Current approaches and future potential for delivering CRISPR/Cas components in oilseeds and millets

The Nucleus, Journal Year: 2024, Volume and Issue: 67(1), P. 141 - 156

Published: April 1, 2024

Language: Английский

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Improving the Genome Editing Efficiency of CRISPR/Cas9 in Melon and Watermelon

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

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Establishing a reliable protoplast system for grapevine: isolation, transformation, and callus induction

PROTOPLASMA, Journal Year: 2025, Volume and Issue: unknown

Published: April 25, 2025

Abstract Protoplasts are single cells enclosed by the plasma membrane after cell wall removal. They widely used in various biotechnological applications, including gene functional analysis, verification of genome editing reagents, and plant regeneration. Recent advances have enabled production non-chimeric transgene-free genome-edited plants using protoplasts. This process involves protoplast isolation, transformation, regeneration, requiring advanced technical skills. Challenges isolation regeneration limited their use editing. In grapevines, however, very few studies reported protoplasts isolated from leaves. Efficient transformation protocols for Chardonnay remain lacking require cultivar-specific optimization. this study, we established a reliable efficient system optimizing conditions PEG-mediated cultivar. The yield viable was approximately 75 × 10 6 per gram leaf material, with viability 91%. A efficiency 87% achieved under optimized conditions. To evaluate ability mesophyll protoplast, transformed untransformed were cultured on solid liquid MS media supplemented 2 mg/L 2,4-D 0.5 BA to facilitate microcalli formation. Microcalli formed feeder layer developed into calli when transferred culture BA. However, unable regenerate roots or shoots. These findings provide foundation further optimization protoplast-based systems potential enhance applications species.

Language: Английский

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Streamlined Protoplast Transfection System for In-vivo Validation and Transgene-free Genome Editing in Banana

Research Square (Research Square), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 6, 2024

The advancement in CRISPR/Cas system has significantly streamlined genome editing plants, rendering it simple, reliable and efficient. However, the development of transgene-free crops is a challenging task for vegetatively propagated plants like banana. In present study, we established banana protoplasts based versatile efficient platform to overcome this limitation. Herein, protocol been optimized protoplast isolation by considering leaf embryogenic cell suspension (ECS) cultivar Grand Naine. Freshly prepared ECS was identified as best source isolation. viability competency were checked transfection with plasmid RNP complex. Polyethylene glycol-mediated using pCAMBIA1302 pJL50TRBO vectors showed GFP expression 30% 70% efficiency, respectively, eventually proving protocol's efficacy. Further, gRNAs targeting β-carotene hydroxylase gene are validated in-vitro cleavage test subsequently used complex formation varied ratios (1:1, 1:2, 1:5 1:10) SpCas9 gRNA1. Among these, 1:2 molar ratio proved generate indel frequency 7%. Sequencing analysis target amplicon revealed mutations upstream PAM region, specifically gRNA1, among three gRNAs. This study evaluated effectiveness in-vivo, yielding inconsistent results that highlight need comprehensive in-vivo validation their functionality. Conclusively, potential be harnessed generation genetically improved

Language: Английский

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Adaptation of bacterial natural single guide RNA (tracr-L) for efficient plant genome editing

Plant Cell Reports, Journal Year: 2024, Volume and Issue: 43(12)

Published: Nov. 23, 2024

Language: Английский

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