Efficient Genome Editing in the Oomycete Phytophthora sojae Using CRISPR/Cas9.
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.
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.
Yufeng
Y
Fang
Center for Genome Research and Biocomputing and Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon.
Linkai
L
Cui
Center for Genome Research and Biocomputing and Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon.
Biao
B
Gu
Center for Genome Research and Biocomputing and Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon.
Felipe
F
Arredondo
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.
Brett M
BM
Tyler
D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D000072669:Gene Editing / Q000379:methods*; D010838:Phytophthora / Q000235:genetics*
D064130:CRISPR-Associated Proteins
CRISPR/Cas9; Phytophthora; gene replacement; genome editing; oomycetes