Fang Yufeng, Cui Linkai, Gu Biao, Arredondo Felipe, Tyler Brett M
Interdisciplinary Ph.D. Program in Genetics, Bioinformatics and Computational Biology, Virginia Tech, Blacksburg, Virginia.
Center for Genome Research and Biocomputing and Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon.
Curr Protoc Microbiol. 2017 Feb 6;44:21A.1.1-21A.1.26. doi: 10.1002/cpmc.25.
Phytophthora is a filamentous fungus-like microorganism, but belongs to the oomycetes, in the kingdom Stramenopila. Phytophthora species are notorious as plant destroyers, causing multibillion-dollar damage to agriculture and natural ecosystems worldwide annually. For a long time, genome editing has been unattainable in oomycetes, because of their extremely low rate of homologous recombination. The recent implementation of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system in the soybean pathogen Phytophthora sojae, an experimental model for oomycetes, has opened up a powerful new research capability for the oomycete community. Here, we describe a detailed protocol for CRISPR/Cas9-mediated genome editing in P. sojae, including single guide RNA (sgRNA) design and construction, efficient gene replacement, and mutant-screening strategies. This protocol should be generally applicable for most culturable oomycetes. We also describe an optimized transformation method that is useful for other Phytophthora spp. including P. capsici and P. parasitica. © 2017 by John Wiley & Sons, Inc.
疫霉是一种丝状真菌样微生物，但属于卵菌纲，属于不等鞭毛总门。疫霉属物种作为植物破坏者而声名狼藉，每年给全球农业和自然生态系统造成数十亿美元的损失。长期以来，由于卵菌的同源重组率极低，基因组编辑在卵菌中一直无法实现。最近，在卵菌的实验模型大豆疫霉中实施了CRISPR/Cas（成簇规律间隔短回文重复序列/CRISPR相关蛋白）系统，为卵菌研究群体开辟了一种强大的新研究能力。在这里，我们描述了一种在大豆疫霉中进行CRISPR/Cas9介导的基因组编辑的详细方案，包括单向导RNA（sgRNA）设计与构建、高效基因替换和突变体筛选策略。该方案通常应适用于大多数可培养的卵菌。我们还描述了一种优化的转化方法，该方法对包括辣椒疫霉和寄生疫霉在内的其他疫霉属物种有用。©2017约翰威立国际出版公司。
