Establishing CRISPR-Cas9 in the sexually dimorphic moss,Ceratodon purpureus DOI Creative Commons
Emilie‐Katherine Tavernier, Emily Lockwood, Pierre‐François Perroud

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Oct. 26, 2023

Abstract The development of CRISPR technologies provides a powerful tool for understanding the evolution and functionality essential biological processes. Here we demonstrate successful CRISPR-Cas9 genome editing in dioecious moss species, Ceratodon purpureus . Using an existing selection system from distantly related hermaphroditic moss, Physcomitrium patens , generated knock-outs APT reporter gene by employing targeted mutagenesis under expression native U6 snRNA promoters. Next, used homology-directed repair (HDR) pathway, combined with CRISPR-Cas9, to knock-in two genes endogenous RPS5A promoter newly developed landing site C. Our results show that molecular tools P. can be extended other mosses across this ecologically important developmentally variable group. These findings pave way precise experiments aimed at identifying genetic basis key functional variation within bryophytes between land plants. Significance Statement We have protocols sexually dimorphic generate knock-ins loci. This work facilitates future genomic fast growing, haploid, suggests these will function much diversity.

Language: Английский

CRISPR-Cas9-mediated deletions of FvMYB46 in Fragaria vesca reveal its role in regulation of fruit set and phenylpropanoid biosynthesis DOI Creative Commons

Arti Rai,

Magne Nordang Skårn,

Abdelhameed Elameen

et al.

BMC Plant Biology, Journal Year: 2025, Volume and Issue: 25(1)

Published: Feb. 25, 2025

Abstract The phenylpropanoid pathway, regulated by transcription factors of the MYB family, produces secondary metabolites that play important roles in fertilization and early phase fruit development. MYB46 factor is a key regulator cell wall structure, lignin flavonoid biosynthesis many plants, but little known about its activity flowers berries F. vesca . For functional analysis FvMYB46, we designed CRISPR-Cas9 construct with an endogenous -specific U6 promoter for efficient specific expression two gRNAs targeting first exon FvMYB46 This generated mutants in-frame 81-bp deletion conserved domain or out-of-frame 82-bp potentially knocking out gene function. In both types mutant pollen germination set were significantly reduced compared to wild type. Transcriptomic revealed positively regulates genes involved processes like xylan metabolism, homeostasis reactive oxygen species (ROS) including biosynthesis. Genes regulating carbohydrate metabolism signalling also deregulated, suggesting might regulate crosstalk between -mutant flowers, flavanol flavan-3-ol contents, especially epicatechin, quercetin-glucoside kaempferol-3-coumaroylhexoside, reduced, observed local reduction content anthers. Together, these results suggest controls fertility biosynthesis, sugar ROS development

Language: Английский

Citations

1
Gene editing tool kit in millets: present status and future directions DOI

Vidhi Sapara,

Mitesh Khisti,

Kalenahalli Yogendra

et al.

The Nucleus, Journal Year: 2024, Volume and Issue: 67(1), P. 157 - 179

Published: April 1, 2024

Language: Английский

Citations

4
An Improved Method for Agrobacterium-Mediated Genetic Transformation of Three Types of Lettuce DOI Creative Commons
M. Roche, Wusheng Liu, Ricardo Hernández

et al.

Plants, Journal Year: 2025, Volume and Issue: 14(4), P. 620 - 620

Published: Feb. 18, 2025

Lettuce genetic transformation is genotype-dependent. In the present study, we have successfully developed an optimized Agrobacterium-mediated protocol for elite lettuce cultivars, which belong to romaine, leaf, and butterhead cultivar types. We type concentration of plant growth regulators (PGRs) selection antibiotics found that use 1-naphthaleneacetic acid (NAA; 0.10 mg/L) 6-benzyladenine (BA; 0.25 as regulators, hygromycin (15 transgenic selection, cotyledons first true leaf explants efficiently yielded transformed plants seven out eleven tested achieving a 24.3–100% efficiency. These cultivars include two romaine-type three leaf-type butterhead-type mark successful romaine ‘Kahu’ ‘Rosalita’, ‘Red Sails’ ‘Royal Oak Leaf’, ‘Lollo Biondo’. also observed substituting with kanamycin (40 resulted in 64.3% efficiency ‘Mariska’, one remaining four cultivars. Our newly protocols are applicable regeneration, enabling or selection.

Language: Английский

Citations

0
Viral-Based Gene Editing System for Nutritional Improvement of Fructan Content in Lettuce DOI Open Access
Yarin Livneh,

Dor Agmon,

Ehud Leor‐Librach

et al.

International Journal of Molecular Sciences, Journal Year: 2025, Volume and Issue: 26(6), P. 2594 - 2594

Published: March 13, 2025

Lettuce is a globally cultivated and consumed leafy crop. Here we developed an efficient tobacco rattle virus (TRV)-based guide RNA (gRNA) delivery system for CRISPR/Cas editing in the commercial lettuce cultivar ‘Noga’. Plants stably expressing Cas9 were inoculated with TRV vectors carrying gRNAs targeting five nutrient-associated genes. The achieved average efficiency of 48.7%, up to 78.9% regenerated plantlets showing independent mutations. This approach eliminates need antibiotic selection, simplifying tissue culture processes. supports diverse applications, including Cas12a large-fragment deletions using dual gRNA sets. Targeting fructan 1-exohydrolase 2 (1-FEH2) gene produced knockout lines significant increases prebiotic dietary fibre content, 5.2-fold, rise degree polymerisation by 2.15 units compared controls. Combining 1-FEH1 1-FEH2 knockouts did not further increase levels, revealing as predominant isozyme lettuce. RT-qPCR analysis showed reduced expression upstream biosynthetic enzyme sucrose:sucrose 1-fructosyl transferase (1-SST), suggesting potential feedback inhibition metabolism. TRV-based approach, utilised here could be applied improve other valuable traits lettuce, may inspire similar systems enhance nutritional content crops.

Language: Английский

Citations

0
A Truncated Endogenous U6 Promoter Enables High-Efficiency CRISPR Editing in Flax (Linum usitatissimum L.) DOI Creative Commons
Feifei Li,

Min Xue,

Dongliang Guo

et al.

Plants, Journal Year: 2025, Volume and Issue: 14(7), P. 1142 - 1142

Published: April 6, 2025

Functional U6 promoters are widely utilized in CRISPR gene editing systems for crops. The identification of endogenous promoter activity and the establishment CRISPR/Cas9 various crops can enhance efficiency accuracy molecular breeding. In this study, four snRNAs were identified genome oil flax (Linum usitatissimum L.) cultivar Longya 10, which exhibit high homology with regions Arabidopsis thaliana snRNA. We cloned constructed fusion expression vectors promoter-driven dual-luciferase reporter genes. Transient transformation Nicotiana benthamiana was performed to measure relative dual luciferase. U6-4 on chromosome 14 showed highest transcriptional activity. Truncations varying lengths from 5' end tested, revealing that a 342 bp fragment possesses an optimal length. Subsequently, we vector LuU6-5P/AtU6-P driving LusPDS sgRNA. Agrobacterium-mediated infection hypocotyls yielded transgenic albino shoots. DNA these shoots used as template amplify fragments, then sequenced. Sequencing analysis revealed using Lu14U6-4-5P achieved higher frequencies at compared AtU6-P-driven systems.

Language: Английский

Citations

0
Isolation and Functional Characterization of a Constitutive Promoter in Upland Cotton (Gossypium hirsutum L.) DOI Open Access
Yang Yang, Xiaorong Li, Chenyu Li

et al.

International Journal of Molecular Sciences, Journal Year: 2024, Volume and Issue: 25(3), P. 1917 - 1917

Published: Feb. 5, 2024

Multiple cis-acting elements are present in promoter sequences that play critical regulatory roles gene transcription and expression. In this study, we isolated the cotton FDH (Fiddlehead) (pGhFDH) using a real-time reverse transcription-PCR (qRT-PCR) expression analysis performed prediction analysis. The plant vector pGhFDH::GUS was constructed Gateway approach used for genetic transformation of Arabidopsis upland plants to obtain transgenic lines. Histochemical staining β-glucuronidase (GUS) activity assay showed GUS protein detected roots, stems, leaves, inflorescences, pods thaliana Notably, high observed different tissues. lines, tissues such as stalks, buds, petals, androecium, endosperm, fibers, where pGhFDH-driven levels were 3–10-fold higher compared those under CaMV 35S at 10–30 days post-anthesis (DPA) during fiber development. results indicate pGhFDH can be an endogenous constitutive drive target genes various facilitate functional genomic studies accelerate molecular breeding.

Language: Английский

Citations

1
Evaluation of two new promoters to express transgenes stably in lettuce (Lactuca sativa L.) DOI
Tatiane Rosa Monteiro, Lídia N. Queiroz, G. B. Cabral

et al.

Plant Cell Tissue and Organ Culture (PCTOC), Journal Year: 2024, Volume and Issue: 157(3)

Published: June 1, 2024

Language: Английский

Citations

1
Establishing CRISPR‐Cas9 in the sexually dimorphic moss, Ceratodon purpureus DOI
Emilie‐Katherine Tavernier, Pierre‐François Perroud, Emily Lockwood

et al.

The Plant Journal, Journal Year: 2024, Volume and Issue: 119(6), P. 2753 - 2764

Published: Aug. 18, 2024

The development of CRISPR technologies provides a powerful tool for understanding the evolution and functionality essential biological processes. Here we demonstrate successful CRISPR-Cas9 genome editing in dioecious moss species, Ceratodon purpureus. Using an existing selection system from distantly related hermaphroditic moss, Physcomitrium patens, generated knock-outs APT reporter gene by employing CRISPR-targeted mutagenesis under expression native U6 snRNA promoters. Next, used homology-directed repair (HDR) pathway, combined with CRISPR-Cas9, to knock two genes endogenous RPS5A promoter newly developed landing site C. Our results show that molecular tools P. patens can be extended other mosses across this ecologically important developmentally variable group. These findings pave way precise experiments aimed at identifying genetic basis key functional variation within bryophytes between land plants.

Language: Английский

Citations

1
Identification of U6 Promoter and Establishment of Gene-Editing System in Larix kaempferi (Lamb.) Carr DOI Creative Commons

Jun-Xia Xing,

Ao-Jie Luo,

Xinhao Wang

et al.

Plants, Journal Year: 2024, Volume and Issue: 14(1), P. 45 - 45

Published: Dec. 26, 2024

This study aimed to establish a CRISPR/Cas9 gene-editing system for Larix kaempferi (Lamb.) Carr. (Japanese larch). We screened L. U6 promoters and used them drive sgRNA expression in the system. The embryogenic callus was as receptor material genetic transformation, frequency types of gene editing were then analyzed. results showed various mutations transgenic materials, including base substitutions deletions, ranged from 5% 14.29%. In summary, we established kaempferi. Our demonstrate that can efficiently edit genes kaempferi, with significantly higher frequencies observed when is driven by endogenous LaU6 compared exogenous promoter ProAtU6-26. work provides technical support functions improvement.

Language: Английский

Citations

1
Cloning and Functional Verification of Endogenous U6 Promoters for the Establishment of Efficient CRISPR/Cas9-Based Genome Editing in Castor (Ricinus communis) DOI Open Access
Masatake Kanai,

Kazumi Hikino,

Shoji Mano

et al.

Genes, Journal Year: 2023, Volume and Issue: 14(7), P. 1327 - 1327

Published: June 23, 2023

Castor (Ricinus communis) seeds are rich in a type of hydroxy fatty acid called ricinoleic acid, which is high demand for the production plant-based plastics, lubricants, and hydraulic oils. However, content ricin, toxic protein, these has restricted further expansion area castor cultivation. Therefore, development ricin-free needed. Genome editing technology, although successfully applied several plant species, still developing stages awaits identification an endogenous U6 promoter with robust function. Here, we searched small nuclear RNA (snRNA) genes genome. This led to six snRNA genes. The promoters were cloned, their function was examined cells using particle delivery method. results showed that length approximately 300 bp from transcription start site sufficient activate gene expression. study provides insights into sequences outlines method verifying plants system.

Language: Английский

Citations

2