CRISPR-Cas12a ribonucleoprotein-mediated gene editing in the plant pathogenic fungus Magnaporthe oryzae

Abstract

Summary Gene replacements through homologous recombination (HR) have been extensively used for functional genomic studies. However, the general efficiency of HR repair can be low in filamentous fungi and the process laborious. Here, we provide a detailed protocol for efficient gene editing by inserting donor DNA into a region of interest following Cas12a ribonucleoprotein (RNP)-mediated DNA double-strand break. We demonstrate this protocol using Magnaporthe oryzae (synonym of Pyricularia oryzae ), a model plant pathogenic fungus that is used to study plant-fungal interactions. For complete details on the use and execution of this protocol, please refer to Huang et al. (2021) .

CRISPR-Cas12a induced DNA double-strand breaks are repaired by multiple pathways with different mutation profiles in Magnaporthe oryzae

“…The fungal cultures were maintained under light at 25 °C on OTA to observe mycelial color change. For high-molecular-weight DNA extraction and protoplast preparation, related mycelial plugs for different strains from OTA were cultured in liquid CM at 28 °C, 120 rpm for 3–4 days as described before 91 , 92 .…”

“…Monarch® RNA Cleanup Kit (New England BioLabs, catalog# T2050L) was used to purify synthesized gRNA after DNase I (RNase-free) treatment (New England BioLabs, catalog# M0303S). 5 μg purified LbCas12a (New England BioLabs, catalog# M0653T or Integrated DNA Technologies, catalog# 10007923) or SpCas9 (New England BioLabs, catalog# M0646M) were incubated with equal molar purified gRNA at 25 °C for 15 min for RNP assembly 91 .…”

“…M. oryzae protoplast preparation was performed as described previously 91 . The fungal mycelium was filtered and dried through 2-layer 6 1/2 inch Disks Non Gauze Milk Filter papers, followed by addition of lysing solution (10 mg/mL Lysing Enzymes from Trichoderma harzianum , Sigma, catalog# L1412-10G, dissolved in 0.7 M NaCl solution) and digestion at 30 °C with 70–80 rpm for 2–3 h in the dark.…”

“…The fungal mycelium was filtered and dried through 2-layer 6 1/2 inch Disks Non Gauze Milk Filter papers, followed by addition of lysing solution (10 mg/mL Lysing Enzymes from Trichoderma harzianum , Sigma, catalog# L1412-10G, dissolved in 0.7 M NaCl solution) and digestion at 30 °C with 70–80 rpm for 2–3 h in the dark. After washing with 1xSTC (20% w/v Sucrose, 50 mM Tris-Hcl pH = 8.0, 50 mM CaCl 2 dissolved in water), the concentration of released protoplasts was adjusted to 8 × 10 6 − 5 × 10 7 protoplasts/mL for transformation 91 .…”

Understanding the Dynamics of Blast Resistance in Rice-Magnaporthe oryzae Interactions

“…In the rice blast fungus M. oryzae , concurrent double editing was accomplished using this approach [ 63 ]. Similarly, Cas12a RNP was successfully used for M. oryzae genome editing in a recent report [ 68 ]. However, there are no reports on CRISPR/Cas-based transformation of M. oryzae for changing pathogenicity or creating novel variants of the pathogen.…”