Ali Zahir, Zaidi Syed Shan-E-Ali, Tashkandi Manal, Mahfouz Magdy M
Laboratory for Genome Engineering, Division of Environmental and Biological Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.
Methods Mol Biol. 2019;2028:167-183. doi: 10.1007/978-1-4939-9635-3_10.
Throughout the world, geminiviruses cause devastating losses in economically important crops, including tomato, cotton, cassava, potato, chili, and cucumber; however, control mechanisms such as genetic resistance remain expensive and ineffective. CRISPR/Cas9 is an adaptive immunity mechanism used by prokaryotes to defend against invading nucleic acids of phages and plasmids. The CRISPR/Cas9 system has been harnessed for targeted genome editing in a variety of eukaryotic species, and in plants, CRISPR/Cas9 has been used to modify or introduce many traits, including virus resistance. Recently, we demonstrated that the CRISPR/Cas9 system could be used to engineer plant immunity against geminiviruses by directly targeting the viral genome for degradation. In this chapter, we describe a detailed method for engineering CRISPR/Cas9-mediated resistance against geminiviruses. This method may provide broad, durable viral resistance, as it can target conserved regions of the viral genome and can also be customized to emerging viral variants. Moreover, this method can be used in many crop species, as it requires little or no knowledge of the host plant's genome.
在全球范围内，双生病毒给包括番茄、棉花、木薯、马铃薯、辣椒和黄瓜在内的经济作物造成了毁灭性损失；然而，诸如遗传抗性等控制机制仍然昂贵且无效。CRISPR/Cas9是原核生物用于抵御噬菌体和质粒入侵核酸的一种适应性免疫机制。CRISPR/Cas9系统已被用于多种真核生物的靶向基因组编辑，在植物中，CRISPR/Cas9已被用于修饰或引入许多性状，包括病毒抗性。最近，我们证明了CRISPR/Cas9系统可通过直接靶向病毒基因组进行降解来构建植物对双生病毒的免疫。在本章中，我们描述了一种构建CRISPR/Cas9介导的对双生病毒抗性的详细方法。这种方法可能提供广泛、持久的病毒抗性，因为它可以靶向病毒基因组的保守区域，也可以针对新出现的病毒变体进行定制。此外，这种方法可用于许多作物物种，因为它几乎不需要或不需要了解宿主植物的基因组。
