Combined Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Detecting Fusarium oxysporum f. sp. cubense Tropical Race 4

Summary

In this protocol, a combined recombinase polymerase amplification CRISPR/Cas12a assay is illustrated for the detection of the invasive plant pathogen, Fusarium oxysporum f. sp. cubense tropical race 4.

Abstract

Regular and accurate surveillance stands central to the efficient management of plant diseases. It can indicate which course of action is most appropriate, and whether prevention, eradication, or no action is required. Surveillance based on symptomology in host plants alone is often not reliable due to similarities in the symptoms caused by biotic and abiotic stresses. Laboratory-based molecular methods such as polymerase chain reaction (PCR) and quantitative (q)PCR are the most commonly and reliably used for plant pathogen detection, but rely on expensive equipment and skilled operators. Here, we describe a protocol combining a simplified DNA extraction, recombinase polymerase amplification (RPA), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (RPA-Cas12a) for the detection of the invasive pathogen, Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). The technique provides a simple single-tube detection alternative that is analytically robust with improved specificity compared to available molecular detection assays and negates the need for expensive and sophisticated laboratory equipment.

Introduction

Fusarium wilt, caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), is globally regarded as one of the most devastating transboundary plant pathogens in history¹. Once introduced into a new area, Foc remains invisible until infected plants develop symptoms. This can take months, allowing the fungus to establish and spread unknowingly. Once established, it is almost impossible to eradicate economically due to the production of resilient chlamydospores and the perennial nature of banana production. The importance of prevention and early detection, thus, is paramount in protecting vulnerable growers¹. The current epidemic of the disease, caused by Foc tropical race 4 (TR4), requires detection by regular surveillance to identify symptomatic banana plants in high-risk areas. Abiotic constraints such as water stress and certain nutrient deficiencies produce external yellowing, which is similar to symptoms produced by Foc TR4 infection. Additionally, symptoms resulting from infection by different Foc races cannot be distinguished. Laboratory-based purification and subsequent phenotypic and molecular-based detection are, therefore, necessary for reliable Foc TR4 detection². Various molecular assays are available for Foc TR4 detection, including PCR, quantitative (q) PCR, and loop-mediated isothermal amplification (LAMP) assays^3,4,5,6,7. Most of these protocols, however, still require expensive and specialized skills and equipment, which must be conducted in laboratory settings. Recombinase polymerase amplification (RPA) can be performed isothermally and, therefore, eliminates the need for such equipment and can be considered for in-field detection protocols⁸.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have increasingly been utilized for improved molecular diagnostics⁹. These systems are employed as they are inexpensive, simple, and do not require the use of special instrumentation, and can therefore be considered for detection in-field. CRISPR-based diagnostics typically involve the isothermal amplification of a target sequence, followed by target recognition via CRISPR-Cas proteins and the collateral cleavage of a DNA or RNA fluorescent reporter dye to indicate the presence of the target^10,11. In CRISPR-Cas12a systems, specifically, CRISPR RNA (crRNA) guides Cas12a to recognize and cleave nucleic acid targets. This crRNA can be designed to target a specific DNA or RNA region of interest through hybridization to a complementary sequence. Once bound, it results in Cas12a cleaving the target with single-nucleotide specificity, followed by collateral cleavage of reporter molecules¹². Recently, CRISPR-Cas12a systems were employed to detect various economically important fungal plant pathogens^{13,14,15,16,17,18,19,20}.

In this protocol, the development of a single-tube detection technique is described, combining a simplified DNA extraction, recombinase polymerase amplification (RPA), and CRISPR/Cas12a technology (RPA-Cas12a) for the detection of Foc TR4, which was visualized both on a portable fluorometer and with the naked eye under an inexpensive light-emitting diode (LED) blue light transilluminator²¹. The method can be further optimized for in-field detection to improve prevention and containment strategies. A similar approach can also be utilized to design Cas12a-based detection assays for other plant pathogens.

Protocol

1. Simplified DNA extraction from plant material

NOTE: See Figure 1 for an overview.

1. Perform DNA extraction.
   1. Transfer 250 mg of each piece of symptomatic pseudostem into a separate extraction bag (see Table of Materials).
   2. Add 2 mL of 0.5 M NaOH-PVP (Table of Materials) to bags with plant material. Grind infected vessels in an extraction bag using a manual grinder like a pestle.
      NOTE: Add 1-2 mL supplementary 0.5 M NaOH-PVP if necessary to get enough liquid extract from the bag.
   3. Add 195 µL of 100 mM Tris-HCL (pH 8.0) (Table of Materials) into 1.5 mL tubes.
   4. Pipette 5 µL of extract from the bag into the 1.5 mL tube with 195 µL of 100 mM Tris-HCL (pH 8.0), diluting the extract 40-fold.
   5. Vortex the tubes for 15-30 s and store at -20 °C until use.

2. crRNA synthesis and quality control

1. Perform transcription.
   1. Using the RNA transcription kit (Table of Materials), add the following to a 0.2 mL PCR tube: 1.5 µL of 10x T7 Reaction buffer, 1.5 µL of each dNTP, 1.5 µL of T7 DNA polymerase mix, 1 µL of nuclease-free water, and 10 µL of 100 µM T7 template DNA for a total volume of 20 µL.
   2. Incubate the tube at 37 °C in a water bath or PCR machine for at least 4 h.
   3. Use a spectrophotometer (Table of Materials) to determine the quantity of transcribed RNA. Dilute the RNA to 250 ng/µL (10 µg) in a volume of 40 µL using nuclease-free water or TE buffer (Supplementary File 1).
      NOTE: The transcribed RNA can be stored at -80 °C until continuing with DNase treatments and cleaning steps.
2. Perform DNase treatment and crRNA cleanup.
   NOTE: Perform all steps at room temperature. Keep RNA and DNase I enzyme on ice.
   1. Treat the RNA sample with DNase I included in the RNA cleaning kit (Table of Materials) according to the manufacturer instructions²².
   2. Clean the RNA sample using the RNA cleaning kit according to manufacturer instructions, following Protocol II²².
   3. Nanodrop the cleaned crRNA to determine the quantity and quality present. Dilute the RNA to 100 ng/µL using nuclease-free water and store at -80 °C until use.
   4. Heat the crRNA with RNA loading dye (Table of Materials) to 70 °C for 10 min in a water bath, and subject the crRNA to electrophoresis on a 1.5% agarose gel to visualize the crRNA synthesized (Figure 2).

3. Recombinase polymerase amplification (RPA)-Cas12a one-tube reaction for in vitro Foc TR4 detection

NOTE: See Figure 3 for an overview.

1. Prepare the RPA and Cas12a reaction mixes.
   NOTE: Ensure that these steps are carried out in a DNA-free space on ice, with the reagents and materials pre-cooled. Wipe the working surface and pipettes with bleach and 70% ethanol before the experiment. See Table 1 for information on all oligonucleotides used in this study.
   1. Prepare a 20 µL Cas12a reaction mixture in a separate 0.5 mL tube (Table of Materials) with the following reagent volumes: 2 µL of 10x NEBuffer r2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 100 µg/mL Recombinant Albumin, pH 7.9), 0.4 µL of 10 µM LbCas12a protein, 0.6 µL of 100 ng/µL crRNA, 0.4 µL of 10 µM reporter, and 16.6 µL of deionized water.
      NOTE: The final concentrations of LbCas12a protein, crRNA, and reporter are 200 nM, respectively.
   2. Prepare a 9.5 µL RPA reaction mixture in a separate 0.5 mL tube with the following reagent volumes: 5 µL of 2x Reaction Buffer (final concentration 1x according to manufacturer guidelines), 1.6 µL of dNTP Mix (final concentration 1.6 mM), 1 µL of 10x Basic E-Mix (final concentration 1x according to manufacturer guidelines), 0.5 µL of each RPA primer at 10 µM (final concentration 0.5 µM each), 0.5 µL of 20x Core Reaction Mix (final concentration 1x according to manufacturer guidelines), and 0.4 µL of deionized water.
   3. Place an 8-tube strip into a tube rack (Table of Materials) on ice.
   4. Transfer the 20 µL Cas12a reaction mix into the tube base.
   5. Transfer the 9.5 µL RPA reaction mix into the tube strip lid. Add 0.5 µL of 280 nM MgOAc (final concentration 14 nM) to the RPA reaction mix in the tube strip lid and mix by pipetting up and down.
   6. Add 0.5 µL of deionized water to the non-template control (NTC) tube lid and close carefully to ensure that the RPA and Cas12a reaction mixtures are not combined.
      NOTE: A non-template control (NTC) is included to ensure reliability of the results.
2. Add template DNA and start the reaction.
   1. Add 0.5 µL of template DNA to the 10 µL RPA reaction mixture containing MgOAc in the tube strip lid, except for the NTC, with deionized water in place of DNA. Always add a known positive control Foc TR4 DNA in one well to ensure the assay worked as intended. Mix by pipetting up and down.
   2. Carefully close the tube strip lids to ensure the RPA and Cas12a reaction does not mix.
   3. Switch on the portable fluorometer (Table of Materials) and create a Run Profile for the RPA-Cas12a assay (Supplementary File 2). Open the saved Run Profile and place the tube strip into the portable fluorometer to incubate for 30 min at 37 °C.
      NOTE: Be careful not to touch the bottom of the tubes when handling the tube strip; instead hold the outer edges at the top left and right end.
   4. Remove the tube strip from the portable fluorometer after 30 min. Invert the tubes six times to mix the RPA and Cas12a reactions and spin down in a mini centrifuge (see Table of Materials) for 5 s.
   5. Place the tube strip back into the portable fluorometer and incubate for a further 30 min at 37 °C using the saved Run Profile.
3. Visualize the results.
   1. Interpret the average relative fluorescence units (RFUs) measured on the portable fluorometer (Supplementary File 2 and the user manual²³).
   2. Visualize the tubes under LED blue light at 465 nm excitation wavelength using a blue light transilluminator (Supplementary File 3). Capture images with a phone camera.

   

Results

Successful detection of Foc TR4 from infected plant material using simplified DNA extraction followed by the combined RPA-Cas12a assay when conducted in the Genie III portable fluorometer is illustrated in Figure 4A. Positive samples show visible fluorescence (Lane 6-8), while negative samples show low endpoint fluorescence (Lane 1-5). When sample tubes were viewed under LED blue light, the Foc TR4-infected samples also showed visible fluorescence (Figure 4B). I...

Discussion

Regular surveillance is an important strategy for effective prevention and containment efforts of Foc TR4 and other important invasive pests. Detection protocols that can be applied in-field, and rapidly and accurately identify pathogens, can fast-track decision-making and limit costs associated with surveillance efforts. To this end, the protocol presented here combined a simplified DNA extraction, conducted directly on sampled plant material, which was subsequently included in a one-tube assay combining RPA and Cas12a ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EnGen Lba Cas12a (Cfp1) Protein	New England Biolabs	M0653T
EnGen Lba Cas12a Diluent	New England Biolabs	B0653A
Eppendorf PCR Tubes	Merck	EP0030124332
Eppendorf Safe-Lock Micro Test Tubes	Merck	EP0030121708
Extraction Bags Universal	BIOREBA	430100
GeneRuler 100 bp DNA Ladder	Thermo Fischer Scientific	SM0241 / SM0242
Genie Centrifuge Mini-6KO	OptiGene	OP-FUGE
Genie III	OptiGene	https://www.optigene.co.uk/instruments/genie-iii/
Genie Strip Holder	OptiGene	GBLOCK-01 / GBLOCK-02 / GBLOCK-03
Genie Strips	OptiGene	OP-0008-50 / OP-0008-500
HiScribe T7 High Yield RNA Synthesis Kit	New England Biolabs	E2040S
NanoDrop ND-1000 UV/Vis Spectrophotometer	Thermo Fischer Scientific	https://caeonline.com/buy/spectrometers/nanodrop-nd-1000/293669253
NEBuffer 3	New England Biolabs	B7003S
NEBuffer r2.1	New England Biolabs	B6002S
Polyvinylpyrrolidone (PVP) powder	Sigma-Aldrich	9003-39-8
RNA Clean and Concentrator-5 Kit	Zymo Research	R1013
RNA Loading Dye	Thermo Fischer Scientific	R0641
SmartBlue Blue Light Transilluminator	Accuris Instruments	E4000-E
Sodium hydroxide (NaOH) pellets	Merck, EMPARTA	K277
Tris (hydroxylmethyl) aminomethane	Merck	77-86-1
TwistAmp Liquid Basic Kit	TwistDx	TALQBAS01