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

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.

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea

“…Delivery of sgRNAs can be achieved either via plasmids or by in vitro synthesized sgRNA. More recently, transformation with Cas9-sgRNA ribonucleoprotein (RNP) complexes has been established in several fungi [23][24][25]. In M. oryzae, a novel selection strategy was developed based on switching between two antagonistic states of resistance (either to the fungicides benomyl or diethofencarb), depending on the sequence of codon 198 of the TUB2 gene, to allow the construction of marker-less mutations [24].…”

Genome editing enables reverse genetics of multicellular development in the choanoflagellateSalpingoeca rosetta

“…The establishment of Cas9-mediated genome editing advances S. rosetta as a model for illuminating the evolution of development in choanoflagellates and their closest living relatives, 245 animals. We were able to overcome initial failed efforts to establish genome editing in S. rosetta by engineering cycloheximide resistance in rpl36a as a selectable marker, similar to the use of selectable markers during the establishment of genome editing in other eukaryotes, including Fungi (Foster et al, 2018), green algae (Ferenczi et al, 2017), and nematodes (Arribere et al, 2014;Kim et al, 2014;Ward, 2015). Single-copy ribosomal protein genes like rpl36a offer 250 certain advantages for engineering drug resistance markers with genome editing.…”

“…2A; . We favored delivering the SpCas9 RNP rather than expressing SpCas9 and gRNAs from plasmids, 130 as overexpressing SpCas9 can be cytotoxic for other organisms (Jacobs et al, 2014;Jiang et al, 2014;Shin et al, 2016;Foster et al, 2018) and RNA polymerase III promoters for driving gRNA expression have not yet been characterized in S. rosetta. After growing transfected cells in the presence of cycloheximide for five days, Sanger sequencing of PCR-amplified rpl36a showed that rpl36a P56Q was the major allele in the population (Fig.…”

“…To overcome this challenge, we sought to simultaneously edit rosetteless and rpl36a by transfecting cells with RNPs complexed with gRNAs and DNA repair templates for both knocking out rosetteless and engineering cycloheximide resistance. In other organisms (Arribere et al, 2014;Kim et al, 2014;Ward, 2015;Foster et al, 2018), this approach has 175 allowed for co-selection by using a selectable marker to improve the recovery of cells that contain a second mutation in a different locus. In S. rosetta, we found that 10.4-16.5% of cycloheximide resistant cells contained the rtls PTS1 allele when rosetteless and rpl36a were coedited ( Fig.…”

Nonproteinaceous effectors: the terra incognita of plant–fungal interactions

“…This combinatorial genetics analysis is promising to identify the set of BGCs collectively involved in a given plant–fungal interaction. These tools are already available in diverse plant pathogenic fungi, including Fusarium oxysporum (Wang et al ., ), Alternaria alternata (Wenderoth et al ., ), Leptosphaeria maculans (Idnurm et al ., ) and P. oryzae (Foster et al ., ). Considering that sRNA effectors seem to target several mechanisms at once, it is expected that functional analyses will lead to strong phenotypes.…”