Efficient Genome Editing in the Oomycete Phytophthora sojae Using CRISPR/Cas9

Yufeng Fang1, Linkai Cui1, Biao Gu1, Felipe Arredondo1, Brett M. Tyler1

1 Center for Genome Research and Biocomputing and Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 21A.1
DOI:  10.1002/cpmc.25
Online Posting Date:  February, 2017
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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.

Keywords: oomycetes; Phytophthora; genome editing; gene replacement; CRISPR/Cas9

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Table of Contents

  • Introduction
  • Basic Protocol 1: sgRNA Design
  • Basic Protocol 2: Preparation of CRISPR/Cas9 Plasmids
  • Basic Protocol 3: Preparation of Homologous Donor Template for HDR‐Mediated Mutation
  • Basic Protocol 4: Optimized P. sojae Transformation
  • Basic Protocol 5: Screening for P. sojae HDR‐Mediated Mutants
  • Support Protocol 1: Small Scale Extraction of P. sojae Genomic DNA (P. sojae gDNA Miniprep)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: sgRNA Design

  • sgRNA design software, EuPaGDT (http://grna.ctegd.uga.edu/)
  • Sequence analysis, FungiDB (http://fungidb.org/)
  • RNA secondary structure prediction, RNA structure (http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html).

Basic Protocol 2: Preparation of CRISPR/Cas9 Plasmids

  • Oligonucleotides (sense and antisense oligonucleotides obtained from an oligonucleotide synthesis service)
  • TE buffer (10 mM Tris·Cl, 1 mM EDTA; pH 8.0)
  • T4 DNA ligase and 10 × buffer (NEB, cat. no. M0202S)
  • Sterile deionized water
  • 0.5 M NaCl
  • pYF515 (“all‐in‐one” plasmid, for expression of both Cas9 and sgRNA)
  • Generalized sgRNA cloning plasmid pYF2.3G‐Ribo‐sgRNA (for two‐plasmid transformation system)
  • Nhe I (NEB, cat. no. R0131S)
  • Bsa I and 10 × CutSmart Buffer (NEB, cat. no. R0535S)
  • PCR purification kit (Qiagen)
  • Chemically competent E. coli DH5α cells
  • Ice
  • LB liquid medium and LB liquid medium containing 100 μg/ml ampicillin (see Elbing and Brent, )
  • SOC liquid medium (see Seidman et al., )
  • LB agar plates containing 100 μg/ml ampicillin (see Elbing and Brent, )
  • Green Taq DNA polymerase and buffer (Genscript)
  • M13 Forward universal primer (5′‐GTTTTCCCAGTCACGACG‐3′)
  • Zyppy plasmid Miniprep Kit (Zymo Research)
  • HiSpeed Plasmid Midi kit (Qiagen)
  • hSpCas9 expression plasmid, pYF2‐PsNLS‐hSpCas9 (for two‐plasmid transformation system)
  • Plasmid Maxi Kit (Qiagen)
  • pYF2‐PsNLS‐hSpCas9‐GFP (for examination of Cas9 expression in situ), optional
  • 37°C incubator‐shaker
  • Thermal cycler
  • 42°C water bath for heat‐shocking cells
  • 1.5‐ml microcentrifuge tubes
  • 0.2‐ml PCR tubes
  • 10‐μl sterile pipet tips for picking colonies from agar plates
  • Flasks for large‐scale culture (100‐ to 250‐ml)
  • Nanodrop microspectrophotometer (http://www.nanodrop.com)
  • 15‐ml bacterial culture tubes
  • Vacuum concentrator
  • Additional reagents and equipment for performing agarose gel electrophoreses (see Voytas, 2000).
The CRISPR plasmids can be obtained from the Tyler Laboratory at Oregon State University upon request.

Basic Protocol 3: Preparation of Homologous Donor Template for HDR‐Mediated Mutation

  • Donor DNA (e.g., mCherry gene)
  • PCR purification kit (Qiagen)
  • Plasmid pBluescript SK II+
  • In‐Fusion HD Cloning kit (Clontech) containing:
  •  5× In‐fusion HD enzyme premix
  • Sterile deionized
  • Ice
  • Chemically competent E. coli DH5α cells
  • LB plates containing 100 μg/ml ampicillin
  • HiSpeed Plasmid Midi kit (Qiagen)
  • Phusion High‐Fidelity DNA Polymerase and Phusion HF Buffer (NEB, cat. no. M0530S)
  • 0.2‐ml PCR tubes
  • Thermal cycler
  • Vacuum concentrator
  • Additional reagents and equipment for E.coli transformation, colony‐PCR and Sanger Sequencing (see protocol 2)

Basic Protocol 4: Optimized P. sojae Transformation

  • Phytophthora sojae race 2 (P6497)
  • V8 liquid and solid medium (see recipe)
  • Nutrient pea broth and agar medium (see recipe)
  • Sterile deionized water
  • 0.8 M mannitol (Sigma, cat. no. M1902), sterilize using a 0.45‐mm filter
  • Enzyme solution (see recipe)
  • W5 solution (see recipe)
  • Ice
  • MMG solution (see recipe)
  • PEG4000 (Fluka, cat. no. 81240)
  • Midiprep of sgRNA expression plasmid (pYF2.3G‐Ribo‐sgRNA, for two‐plasmid system; see protocol 2)
  • Maxiprep of Cas9 expression plasmid (pYF2‐hSpCas9, for two‐plasmid system; see protocol 2) or
  • Midiprep of “all‐in‐one” Cas9‐sgRNA expression plasmid (pYF515, for single‐plasmid system; see protocol 2)
  • Midiprep of homologous donor plasmid (see protocol 3)
  • Regeneration medium (see recipe)
  • Ampicillin (Sigma)
  • G418 (Geneticin, AG Scientific)
  • PEG‐calcium transformation solution (see recipe)
  • #3 Cork borer (6‐mm diameter) or scalpels
  • 250‐ml Erlenmeyer flasks with cotton stoppers
  • Cotton stoppers
  • Aluminum foil
  • 25°C incubator
  • Miracloth (EMD Millipore, cat. no. 475855‐1R)
  • 25‐mm deep petri dishes
  • Two stainless steel laboratory spatulas
  • Rotary shaker
  • Light microscope (inverted format is more convenient)
  • 70‐μm Falcon nylon mesh cell strainer (BD Biosciences, cat. no. 08‐771‐2)
  • Beckman Coulter benchtop centrifuge with swing buckets
  • 50‐ml Falcon tubes
  • 1.5‐ml microcentrifuge tubes
  • 90‐mm × 15‐mm petri dishes
  • Benchtop centrifuge
  • Hot bead sterilizers or bunsen burner (used for sterilization of the scalpels)
  • 3‐ml plastic transfer pipets

Basic Protocol 5: Screening for P. sojae HDR‐Mediated Mutants

  • P. sojae transformants growing on nutrient pea agar ( protocol 4, step 28b)
  • V8 liquid medium (see recipe)
  • G418 (Geneticin, AG Scientific)
  • Phusion High‐Fidelity DNA polymerase and Phusion HF Buffer (NEB, cat. no. M0530S)
  • PCR purification kit (Qiagen)
  • 12‐well tissue culture plates
  • 25°C and 14°C incubators
  • Fluorescence microscope
  • Thermal cycler
  • Parafilm
  • Hemacytometer
  • Additional reagents and equipment for obtaining P. sojae gDNA by miniprep (see the Support*Protocol)

Support Protocol 1: Small Scale Extraction of P. sojae Genomic DNA (P. sojae gDNA Miniprep)

  • Regenerated P. sojae hyphae mixture (e.g. from protocol 4, step 22) or P. sojae individual transformants growing in V8 liquid medium (e.g. from protocol 5, step 1)
  • Acid‐treated 0.5‐mm glass beads (Sigma)
  • P. sojae lysis buffer (see recipe)
  • 10 mg/ml RNase A, from bovine pancreas (Sigma, cat. no. R4875)
  • Ice
  • Liquid nitrogen
  • Phenol‐chloroform‐isoamyl alcohol (25:24:1, saturated with 10 mM Tris·Cl, pH 8.0, 1 mM EDTA; Sigma, Cat. no. P3803)
  • Isopropanol
  • 70% (v/v) ethanol
  • Sterile deionized water
  • 2‐ and 1.5‐ml microcentrifuge tubes
  • Benchtop centrifuge
  • Vortex mixer
  • Sterile 200‐µl tips or toothpicks
  • Kimwipes
  • Pellet pestle (disposable polypropylene pestles)
  • 37°C incubator
  • Refrigerated benchtop centrifuge
  • Vacuum concentrator
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