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

. 2021 Dec 24;3(1):101072.


doi: 10.1016/j.xpro.2021.101072.


eCollection 2022 Mar 18.

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Jun Huang et al.


STAR Protoc.


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Abstract

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).


Keywords:

Biotechnology and bioengineering; CRISPR; Genomics; Microbiology; Model Organisms; Molecular Biology; Plant sciences.

Conflict of interest statement

The authors declare no competing interests.

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Graphical abstract


Figure 1



Figure 1

Schematic diagram of Cas12a cutting Blue line highlights the PAM sequence, the orange line indicates the designed targeting sequence. Arrows indicate the cutting site (17th base after PAM in the non-targeted strand and 22nd base after PAM in the targeted strand) of Cas12a, which is represented by the purple shape. The gRNA contains a hairpin at the 5′ end to facilitate Cas12a interaction.


Figure 2



Figure 2

Schematic examples of oligo design


Figure 3



Figure 3

Different protoplast status after lysing enzyme digestion (A–C) (A) An example of healthy protoplasts, (B) undigested mycelium, and (C) collapsed protoplasts each highlighted by black circles. Scale Bars = 50 μm


Figure 4



Figure 4

Schematic illustration of PCR primer pairs used for genotyping


Figure 5



Figure 5

Representative phenotyping and genotyping outcomes at BUF1 locus with BUF1-guide1 RNP and no-homology HYG DNA donor (A) Δbuf1 showed the buff/orange mycelial color in OTA. CP641 (buf-) derived from O-137 is the positive strain for buff mycelia. O-137 (BUF+) is the wild-type strain used in this assay. (B) DNA extracted from the transformants in (A) were used for genotyping with BUF1, BU1 5′upstream, BUF1 3′ downstream and Actin primer pairs (loading control). – and + indicated the water (negative control) and O-137 genomic DNA (positive control) used in PCR reaction.

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