Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton
Abstract Background Upland 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 CRIS...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2021-02-01
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| Series: | Plant Methods |
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| Online Access: | https://doi.org/10.1186/s13007-021-00712-x |
| _version_ | 1818408620432293888 |
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| author | Mohamed Ramadan Muna Alariqi Yizan Ma Yanlong Li Zhenping Liu Rui Zhang Shuangxia Jin Ling Min Xianlong Zhang |
| author_facet | Mohamed Ramadan Muna Alariqi Yizan Ma Yanlong Li Zhenping Liu Rui Zhang Shuangxia Jin Ling Min Xianlong Zhang |
| author_sort | Mohamed Ramadan |
| collection | DOAJ |
| description | Abstract Background Upland 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. Results To 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. Conclusion All 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. |
| first_indexed | 2024-12-14T09:46:38Z |
| format | Article |
| id | doaj.art-4d590bdfce61449fbd3b65e621bc5958 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-14T09:46:38Z |
| publishDate | 2021-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-4d590bdfce61449fbd3b65e621bc59582022-12-21T23:07:36ZengBMCPlant Methods1746-48112021-02-0117111310.1186/s13007-021-00712-xEfficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cottonMohamed Ramadan0Muna Alariqi1Yizan Ma2Yanlong Li3Zhenping Liu4Rui Zhang5Shuangxia Jin6Ling Min7Xianlong Zhang8National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural UniversityNational Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural UniversityNational Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural UniversityNational Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural UniversityNational Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural UniversityNational Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural UniversityNational Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural UniversityNational Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural UniversityNational Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural UniversityAbstract Background Upland 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. Results To 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. Conclusion All 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.https://doi.org/10.1186/s13007-021-00712-xCottonCRISPR/Cas9Genome editingMale sterilityPooled sgRNAs assembly |
| spellingShingle | Mohamed Ramadan Muna Alariqi Yizan Ma Yanlong Li Zhenping Liu Rui Zhang Shuangxia Jin Ling Min Xianlong Zhang Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton Plant Methods Cotton CRISPR/Cas9 Genome editing Male sterility Pooled sgRNAs assembly |
| title | Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton |
| title_full | Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton |
| title_fullStr | Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton |
| title_full_unstemmed | Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton |
| title_short | Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton |
| title_sort | efficient crispr cas9 mediated pooled sgrnas assembly accelerates targeting multiple genes related to male sterility in cotton |
| topic | Cotton CRISPR/Cas9 Genome editing Male sterility Pooled sgRNAs assembly |
| url | https://doi.org/10.1186/s13007-021-00712-x |
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