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

Overexpression of grape ABA receptor gene VaPYL4 enhances tolerance to multiple abiotic stresses in Arabidopsis DOI Creative Commons
Chong Ren, Yangfu Kuang,

Yanping Lin

et al.

BMC Plant Biology, Journal Year: 2022, Volume and Issue: 22(1)

Published: June 2, 2022

Abscisic acid (ABA) plays a crucial role in abiotic stress responses. The pyrabactin resistance (PYR)/PYR-like (PYL)/regulatory component of ABA receptor (RCAR) proteins that have been characterized as receptors function the core components signaling pathway. However, functions grape PYL genes response to different stresses, particularly cold stress, remain less studied.In this study, we investigated expression profiles upon treatment and isolated VaPYL4 gene from Vitis amurensis, cold-hardy species. Overexpression calli Arabidopsis resulted enhanced tolerance. Moreover, plant drought salt was also improved by overexpressing Arabidopsis. More importantly, evaluated contribution growth development after with cold, simultaneously. transgenic plants showed higher survival rates, earlier flowering phenotype, heavier fresh weight seedlings siliques when compared wild-type plants. Physiological analyses had much lower content malondialdehyde (MDA) peroxidase (POD) activity. Stress-responsive such RD29A (Responsive desiccation 29A), COR15A (Cold responsive 15A) KIN2 (Kinase 2) were significantly up-regulated VaPYL4-overexpressing plants.Our results show overexpression could improve performance which therefore provides useful strategy for engineering future crops deal adverse environments.

Language: Английский

Citations

34
Highly efficient activation of endogenous gene in grape using CRISPR/dCas9-based transcriptional activators DOI Creative Commons
Chong Ren, Huayang Li, Yanfei Liu

et al.

Horticulture Research, Journal Year: 2022, Volume and Issue: 9

Published: Jan. 1, 2022

Abstract Overexpression and knockout (or knockdown) of gene interest are two commonly used strategies for functional study. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system-mediated has been applied in most plant species, including grapevine. However, CRISPR/dCas9 (deactivated Cas9)-based transcriptional activation is still unreported fruit crops, although a few studies have documented Arabidopsis rice. Here, we tested activators VP64 TV endogenous genes grape. Both the dCas9-VP64 dCas9-TV systems efficient enough UDP-glucose flavonoid glycosyltransferases (UFGT) grape cells. The effectiveness system UFGT was about 1.6- to 5.6-fold, while efficiency around 5.7- 7.2-fold. Moreover, grapevine plants, highly cold-responsive transcription factor CBF4 achieved by using system. expression increased 3.7- 42.3-fold transgenic plants. Compared with wild-type CBF4-activated plants exhibited lower electrolyte leakage after cold treatment. Our results demonstrate grape, which will facilitate application this economically important species.

Language: Английский

Citations

30
CRISPR/Cas9 technology and its application in horticultural crops DOI Creative Commons
Yang Liu,

Chunling Zhang,

Xiao‐Fei Wang

et al.

Horticultural Plant Journal, Journal Year: 2022, Volume and Issue: 8(4), P. 395 - 407

Published: April 29, 2022

Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9 (CRISPR/Cas9) system has recently become one popular technology due to its efficiency, precision, and simplicity compared with other genome editing tools such as Zinc Finger Nucleases (ZFNs) Transcription Activator Like Effector (TALENs). Horticultural crops provide energy health-keeping nutrients humankind. Genome-editing widely adopted in horticultural breeding the increasing demand for high yield better-quality crops. Here, we describe CRISPR/Cas9 construction, optimization, including sgRNA promoter, design, Cas9 protein SpCas9 variants orthologs, vector delivery methods. We also summarized application of this plants stress responses enhancement, fruit quality improvement, cultivation traits modification. This detailed review was compiled help establish comprehensive understanding systems a reference further developing manipulate plant effectively.

Language: Английский

Citations

29
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

19
Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding DOI Creative Commons
Junming Zhou,

Xinchao Luan,

Yixuan Liu

et al.

Plants, Journal Year: 2023, Volume and Issue: 12(7), P. 1478 - 1478

Published: March 28, 2023

Following recent developments and refinement, CRISPR-Cas9 gene-editing technology has become increasingly mature is being widely used for crop improvement. The application of CRISPR/Cas9 enables the generation transgene-free genome-edited plants in a short period advantages simplicity, high efficiency, specificity, low production costs, which greatly facilitate study gene functions. In plant molecular breeding, efficiency system proven to be key step influencing effectiveness with improvements recently becoming focus reported scientific research. This review details strategies methods improving editing including Cas9 variant enzyme engineering, effect multiple promoter driven Cas9, gRNA efficient optimization expression strategies. It also briefly introduces CRISPR/Cas12a BE PE precision editing. These are beneficial further development systems field breeding.

Language: Английский

Citations

19
Targeted mutagenesis with sequence‐specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects DOI Creative Commons

David May,

Katalin Paldi,

Fredy Altpeter

et al.

The Plant Genome, Journal Year: 2023, Volume and Issue: 16(2)

Published: Jan. 24, 2023

Many of the world's most important crops are polyploid. The presence more than two sets chromosomes within their nuclei and frequently aberrant reproductive biology in polyploids present obstacles to conventional breeding. a larger number homoeologous copies each gene makes random mutation breeding daunting task for polyploids. Genome editing has revolutionized improvement polyploid as multiple and/or alleles can be edited simultaneously while preserving key attributes elite cultivars. Most genome-editing platforms employ sequence-specific nucleases (SSNs) generate DNA double-stranded breaks at target gene. Such typically repaired via error-prone nonhomologous end-joining process, which often leads frame shift mutations, causing loss function. enhanced disease resistance, yield components, end-use quality crops. However, identification candidate targets, genotyping, requirement high mutagenesis efficiency remain bottlenecks targeted In this review, we will survey tremendous progress SSN-mediated crop improvement, discuss its challenges, identify optimizations needed sustain further progress.

Language: Английский

Citations

16
CRISPR/Cas9-based gene activation and base editing in Populus DOI Creative Commons
Tao Yao, Guoliang Yuan, Haiwei Lu

et al.

Horticulture Research, Journal Year: 2023, Volume and Issue: 10(6)

Published: May 2, 2023

The genus

Language: Английский

Citations

16
Genomic Designing for Biotic Stress Resistant Grapevine DOI
Silvia Vezzulli, David Gramaje, Javier Tello

et al.

Springer eBooks, Journal Year: 2022, Volume and Issue: unknown, P. 87 - 255

Published: Jan. 1, 2022

Language: Английский

Citations

23
Applications of CRISPR/Cas genome editing in economically important fruit crops: recent advances and future directions DOI Creative Commons

Zhimin Ma,

Lijing Ma, Junhui Zhou

et al.

Molecular Horticulture, Journal Year: 2023, Volume and Issue: 3(1)

Published: Jan. 28, 2023

Fruit crops, consist of climacteric and non-climacteric fruits, are the major sources nutrients fiber for human diet. Since 2013, CRISPR/Cas (Clustered Regularly Interspersed Short Palindromic Repeats CRISPR-Associated Protein) genome editing system has been widely employed in different plants, leading to unprecedented progress genetic improvement many agronomically important fruit crops. Here, we summarize latest advancements including efforts decipher mechanisms behind plant development immunity, We also highlight potential challenges improvements application tools optimizing expression cassette, improving delivery efficiency reagents, increasing specificity editing, transformation regeneration system. In addition, propose perspectives on crop breeding especially crops challenges. It is worth noting that manipulate with systems urgently needed demonstration.

Language: Английский

Citations

14
Enhancement of the CRISPR/Cas9-Based Genome Editing System in Lettuce (Lactuca sativa L.) Using the Endogenous U6 Promoter DOI Creative Commons

Young-Sun Riu,

Gwang Hoon Kim, Ki Wha Chung

et al.

Plants, Journal Year: 2023, Volume and Issue: 12(4), P. 878 - 878

Published: Feb. 15, 2023

The CRISPR/Cas9 system has been widely applied as a precise gene-editing tool for studying gene functions well improving agricultural traits in various crop plants. Here, we optimized lettuce (Lactuca sativa L.) using the endogenous U6 promoter and proved that PHOT2 is versatile target gene. We isolated LsU6-10 from 10 snRNA genes identified genome database comparison with AtU6-26 used to drive sgRNAs lettuce. Two vectors were constructed promoters sgRNA361 chloroplast avoidance response was defective lettuces biallelic mutations targeted gene, Arabidopsis phot2 mutant. stably heritable R0 R2 generations, high efficiency enabled selection of transgene-free lines R1 generation establishment independent mutants generation. Our results suggest more effective than driving sgRNA useful verify editing without any detrimental effects on plant growth, which often consideration conventional genes.

Language: Английский

Citations

14