Mohamed Ramadan1,2, Muna Alariqi1, Yizan Ma1, Yanlong Li1, Zhenping Liu1, Rui Zhang1, Shuangxia Jin1, Ling Min3, Xianlong Zhang1. 1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, Hubei, China. 2. Department of Plant Genetic Resources, Division of Ecology and Dry Land Agriculture, Desert Research Center, Cairo, Egypt. 3. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, Hubei, China. lingmin@mail.hzau.edu.cn.
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.
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.
Entities:
Keywords:
CRISPR/Cas9; Cotton; Genome editing; Male sterility; Pooled sgRNAs assembly
Authors: Z Jeffrey Chen; Brian E Scheffler; Elizabeth Dennis; Barbara A Triplett; Tianzhen Zhang; Wangzhen Guo; Xiaoya Chen; David M Stelly; Pablo D Rabinowicz; Christopher D Town; Tony Arioli; Curt Brubaker; Roy G Cantrell; Jean-Marc Lacape; Mauricio Ulloa; Peng Chee; Alan R Gingle; Candace H Haigler; Richard Percy; Sukumar Saha; Thea Wilkins; Robert J Wright; Allen Van Deynze; Yuxian Zhu; Shuxun Yu; Ibrokhim Abdurakhmonov; Ishwarappa Katageri; P Ananda Kumar; Yusuf Zafar; John Z Yu; Russell J Kohel; Jonathan F Wendel; Andrew H Paterson Journal: Plant Physiol Date: 2007-12 Impact factor: 8.340