CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus

Foster, Andrew J., Martin-Urdiroz, Magdalena, Yan, Xia, Wright, Harriet Sabrina, Soanes, Darren M. and Talbot, Nicholas J. (2018) CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus. Scientific Reports, 8. ISSN 2045-2322

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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.

Item Type: Article
Uncontrolled Keywords: beta-tubulin gene,magnaporthe-grisea,saccharomyces-cerevisiae,human-cells,genome,resistance,system,cas9,identification,endonuclease
Faculty \ School:
Depositing User: LivePure Connector
Date Deposited: 15 Feb 2019 09:30
Last Modified: 18 Mar 2020 02:39
URI: https://ueaeprints.uea.ac.uk/id/eprint/69934
DOI: 10.1038/s41598-018-32702-w

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