Abstract
The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species.
Highlights
In recent years, the use of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided Cas[9] endonuclease, has facilitated genome editing technologies and become the leading tool used to generate specific changes to DNA sequences in a wide range of species[1]
We report a ribonucleoprotein-CRISPR-Cas[9] (RNP-CRISPR-Cas9) system for genome editing in M. oryzae, using purified nuclear-localised Cas[9] (Cas9-NLS) and in vitro synthesised small guide RNAs (sgRNA), an approach pioneered in Caenorhabditis elegans[15]
Arazoe and co-workers reported successful generation of rsy[1] mutants in M. oryzae using CRISPRCas[9], it is likely that their constructs were only transiently present in the cell[14]
Summary
The use of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided Cas[9] endonuclease, has facilitated genome editing technologies and become the leading tool used to generate specific changes to DNA sequences in a wide range of species[1]. We report a ribonucleoprotein-CRISPR-Cas[9] (RNP-CRISPR-Cas9) system for genome editing in M. oryzae, using purified nuclear-localised Cas[9] (Cas9-NLS) and in vitro synthesised sgRNA, an approach pioneered in Caenorhabditis elegans[15]. This procedure generates highly efficient rates of mutation in M. oryzae at a genomic target sequence, when a donor DNA carrying a selectable marker sequence and capable of repairing the DSB is co-transformed with the RNP into fungal protoplasts. We have employed a novel selection strategy that exploits negative cross resistance to two fungicides to enable CRISPR mediated counterselection This counter-selection method allows mutants to be created that are isogenic to an original wild-type strain. We believe that using RNP-CRISPR-Cas[9] will permit precise and rapid gene manipulation in M. oryzae and other fungi, and thereby accelerate the pace of research in this economically important plant pathogen
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