ArticlePlant methods2021
Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton.
Article in Plant methods, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 23 papers.
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Who cites it
23 citing papers in PubMed, 45 citations in OpenAlex.
- A CRISPR-Cas9 library to target putative redundant gene sets facilitates their functional exploration in grain development in rice.BMC plant biology · 2025Article
- Enhancing Specificity, Precision, Accessibility, Flexibility, and Safety to Overcome Traditional CRISPR/Cas Editing Challenges and Shape Future Innovations.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2025Review
- Deciphering the dynamic expression network of fiber elongation and the functional role of the GhTUB5 gene for fiber length in cotton based on an introgression population of upland cotton.Journal of advanced research · 2025Article
- Transdermal delivery of CRISPR/Cas9-mediated melanoma gene therapy via polyamines-modified thermosensitive hydrogels.Journal of nanobiotechnology · 2025Article
- Article
- Plant Functional Genomics Based on High-Throughput CRISPR Library Knockout Screening: A Perspective.Advanced genetics (Hoboken, N.J.) · 2024Article
- Advances in genome sequencing and artificially induced mutation provides new avenues for cotton breeding.Frontiers in plant science · 2024Review
- CRISPR/Cas9-based mutant library screening: the discovery of novel genes regulating immune responses in cotton and rice.Frontiers in plant science · 2024Review
- Construction of Host Plant Insect-Resistance Mutant Library by High-Throughput CRISPR/Cas9 System and Identification of A Broad-Spectrum Insect Resistance Gene.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2024Article
- The 4Fs of cotton: genome editing of cotton for fiber, food, feed, and fuel to achieve zero hunger.Frontiers in genome editing · 2024Review
- A comprehensive overview of cotton genomics, biotechnology and molecular biological studies.Science China. Life sciences · 2023Review
- Targeted mutagenesis with sequence-specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects.The plant genome · 2023Review
- High-temperature stress in crops: male sterility, yield loss and potential remedy approaches.Plant biotechnology journal · 2023Review
- Multi-Knock-a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants.Nature plants · 2023Article
- BREEDIT: a multiplex genome editing strategy to improve complex quantitative traits in maize.The Plant cell · 2023Article
- Improved cotton yield: Can we achieve this goal by regulating the coordination of source and sink?Frontiers in plant science · 2023Review
- Current insights and advances into plant male sterility: new precision breeding technology based on genome editing applications.Frontiers in plant science · 2023Review
- Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant.Cells · 2022Article
- CRISPR/Cas9-mediated saturated mutagenesis of the cottonPlant direct · 2022Article
- Ribozyme-mediated CRISPR/Cas9 gene editing in pyrethrum (Tanacetum cinerariifolium) hairy roots using a RNA polymerase II-dependent promoter.Plant methods · 2022Article
Corrections and comments
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Authors and funding
9 authors at 1 institution in 2 countries.
Funding
Abstract
backgroundUpland cotton (Gossypium hirsutum), harboring a complex allotetraploid genome, consists of A and D sub-genomes. Every gene has multiple copies with high sequence similarity that makes genetic, genomic and functional analyses extremely challenging. The recent accessibility of CRISPR/Cas9 tool provides the ability to modify targeted locus efficiently in various complicated plant genomes. However, current cotton transformation method targeting one gene requires a complicated, long and laborious regeneration process. Hence, optimizing strategy that targeting multiple genes is of great value in cotton functional genomics and genetic engineering.
resultsTo target multiple genes in a single experiment, 112 plant development-related genes were knocked out via optimized CRISPR/Cas9 system. We optimized the key steps of pooled sgRNAs assembly method by which 116 sgRNAs pooled together into 4 groups (each group consisted of 29 sgRNAs). Each group of sgRNAs was compiled in one PCR reaction which subsequently went through one round of vector construction, transformation, sgRNAs identification and also one round of genetic transformation. Through the genetic transformation mediated Agrobacterium, we successfully generated more than 800 plants. For mutants identification, Next Generation Sequencing technology has been used and results showed that all generated plants were positive and all targeted genes were covered. Interestingly, among all the transgenic plants, 85% harbored a single sgRNA insertion, 9% two insertions, 3% three different sgRNAs insertions, 2.5% mutated sgRNAs. These plants with different targeted sgRNAs exhibited numerous combinations of phenotypes in plant flowering tissues.
conclusionAll targeted genes were successfully edited with high specificity. Our pooled sgRNAs assembly offers a simple, fast and efficient method/strategy to target multiple genes in one time and surely accelerated the study of genes function in cotton.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.