Ramadan Mohamed, Alariqi Muna, Ma Yizan, Li Yanlong, Liu Zhenping, Zhang Rui, Jin Shuangxia, Min Ling, Zhang Xianlong
National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
Department of Plant Genetic Resources, Division of Ecology and Dry Land Agriculture, Desert Research Center, Cairo, Egypt.
Plant Methods. 2021 Feb 8;17(1):16. doi: 10.1186/s13007-021-00712-x.
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.
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.
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.
陆地棉（Gossypium hirsutum）具有复杂的异源四倍体基因组，由A和D亚基因组组成。每个基因都有多个具有高度序列相似性的拷贝，这使得遗传、基因组和功能分析极具挑战性。CRISPR/Cas9工具的近期可用性提供了在各种复杂植物基因组中有效修饰目标位点的能力。然而，目前针对单个基因的棉花转化方法需要复杂、漫长且费力的再生过程。因此，针对多个基因的优化策略在棉花功能基因组学和基因工程中具有重要价值。
为了在单个实验中靶向多个基因，通过优化的CRISPR/Cas9系统敲除了112个与植物发育相关的基因。我们优化了混合sgRNA组装方法的关键步骤，通过该方法将116个sgRNA汇集到4组中（每组由29个sgRNA组成）。每组sgRNA在一个PCR反应中进行编译，随后经过一轮载体构建、转化、sgRNA鉴定以及一轮遗传转化。通过农杆菌介导的遗传转化，我们成功获得了800多株植物。对于突变体鉴定，使用了下一代测序技术，结果表明所有生成的植物均为阳性，且所有目标基因均被覆盖。有趣的是，在所有转基因植物中，85%含有单个sgRNA插入，9%含有两个插入，3%含有三种不同的sgRNA插入，2.5%含有突变的sgRNA。这些具有不同靶向sgRNA的植物在植物开花组织中表现出多种表型组合。
所有目标基因均成功以高特异性进行了编辑。我们的混合sgRNA组装提供了一种简单、快速且高效的方法/策略，可一次性靶向多个基因，并肯定加速了棉花基因功能的研究。
