Detection and validation of the crossover type HR in the genome of P. oryzae using reporter gene constructs

We previously established an HR detection/selection system for evaluating the DSB-mediated HR in P. oryzae21. This system comprises two nonfunctional yellow fluorescent protein (YFP) and blasticidin S (BS) deaminase (BSD) fusion genes (ISTG-YFP::BSD and RS-YFP::BSD) and a rare-cutting endonuclease I-SceI. To validate whether the crossover type HR can be induced by targeted DSBs in the genome of P. oryzae, we further developed this system by modifying pRS-YFP::BSD, for detecting the crossover type HR. The bialaphos resistant (bar) gene cassette was inserted into the pRS-YFP::BSD vector, which resulted in pRS-YFP::BSD-bar (donor vector) (Fig. 1a). The donor and I-SceI expression (DSB inducer) vectors were simultaneously introduced into the protoplasts of P. oryzae transformant ISTG6 that has a single copy of ISTG-YFP::BSD (recipient gene) in the genome22. Since the recipient gene contains an 18-bp I-SceI recognition site between 327 and 328 bp of the YFP::BSD open reading frame, co-introduction with the DSB inducer and donor vector can induce the recipient gene specific DSB and HR between the donor and recipient YFP::BSD gene regions (Fig. 1a). The HR occurred-cells would include functionally restored YFP::BSD gene. When the gene conversion type HR occurred between the donor and recipient genes, the cells would reveal only blasticidin S resistance. In contrast, the crossover type HR would induce the integration of the whole donor vector sequence into the genome, which would result in double-resistant colonies for BS and bialaphos (Fig. 1a). The transformed protoplasts were selected by BS, and then the obtained colonies were counted and transferred onto PSA medium containing bialaphos (30 µg/mL). Consistent with the previous study, co-introduction with the DSB inducer and donor template generated BS resistant colonies; however, no drug-resistant colonies were obtained when only the donor vector was introduced into protoplasts (Fig. 1b). About a half of the BS resistant colonies showed resistance against bialaphos (Fig. 1b). PCR and sequencing analysis showed that randomly selected double drug resistant colonies (5/5) were produced by crossover type HR. Similar results were observed when the protoplasts were initially selected by bialaphos, and then the colonies were transferred onto the PSA plates with BS. Nevertheless, the double-resistant colonies were fewer than those initially selected by BS (Fig. 1b), suggesting that the initially selected bialaphos resistant colonies comprised the transformants produced by the ectopic insertion of bar cassette. These results indicated that introducing site-specific DSB could efficiently induce crossover type HR at the recipient gene locus in P. oryzae.

Single crossover-mediated targeted nucleotide substitution with CRISPR/Cas9 system

We previously succeeded in increasing the HR-mediated targeted gene replacement at the scytalone dehydratase (SDH) gene (MGG_05059) locus via cotransfromation with the fungal CRISPR/Cas9 and targeting vectors23. The SDH gene disrupted mutants can be detected by the loss of melanin deposition (white phenotype). To induce and evaluate the single crossover-mediated target base substitutions, we constructed a donor vector optimized for the induction of single crossover type HR. The 1335-bp SDH region containing point mutations at the CRISPR/Cas9 target site was obtained by fusion PCR, and was cloned into the vector harboring the hygromycin B phosphotransferase (hph) gene cassette (pMK-PSDH). Because the DNA strand exchanges occur after the DSB repair of cleaved DNA by the copy of the donor DNA sequence, specific DSB-mediated single crossover would induce a pin-point base substitution (stop codon insertion) at the desired SDH locus and integrate whole vector sequence containing hph gene cassette into the genome (Fig. 2b). In contrast, the gene conversion type HR would occur only in the base substitutions (Fig. 2b). The donor vector and each pCRISPR/Cas-U6-1 site2, pCRISPR/Cas-U6-2 site2, or pCRISPR/Cas-TrpC site2, targeting SDH gene23, were simultaneously introduced into the wild-type protoplasts and we obtained the hygromycin B resistant colonies. By introducing with the donor and CRISPR/Cas9 vector, the white colony count was dramatically increased, compared with that of only the donor vector (Fig. 2c,d). Sequencing analysis showed that all the white colonies (5/5) comprised desired point mutations (Fig. 2e) and the whole vector sequences were integrated into the SDH locus. Consistent with the previous study23, in these white colonies, the Cas9 gene was not detected by the PCR analysis using a specific primer set (Table S1). These results indicated that a single crossover-mediated HR was induced by CRISPR-based DSB and this strategy enables targeted base substitutions with high efficiency.

Figure 2 Single crossover-mediated targeted nucleotide substitution of the scytalone dehydratase gene (SDH) with CRISPR/Cas9. (a) Schematic representation of pCRISPR/Cas vector and its products for the target DNA cleavage. The expressed Cas9 and single-guide (sg) RNA from the pCRISPR/Cas vector form ribonucleoprotein (RNP) complex in the fungal cell. The RNP cleaves the sgRNA/DNA hybrid sequence by catalyzing nickase domain, RuvC, and HNH. The effective cleavage needs the correct protospacer-adjacent motif (PAM: NGG) that follows the target sequence in the complementary DNA strand. Pro.: promoter, Term.: terminator, NLS: nuclear localization signal. (b) Schematic representation of the single crossover-mediated SDH disruption. The CRISPR/Cas9 target sequence of SDH homologous region in pMK-PSDH was modified to evade the CRISPR/Cas9 cleavage and was introduced with a stop codon. hph: hygromycin B phosphotransferase. (c) Melanin depositions in the wild type (left) and SDH disrupted transformants (white colony) (right). (d) The efficiencies of the single crossover-mediated SDH gene disruption. Total colony: number of hygromycin B-resistant colonies obtained from triplicate experiments. White colony: number of hygromycin B-resistant colonies presenting the white phenotype. Efficiency: percentage of total white colonies. (e) Sequences of the CRISPR/Cas9 target region in the wild type, donor vector, and white colony of the transformants. Full size image

To assess the feasibility of this strategy, the donor vectors harboring very short homologous sequence (1000, 750, 500, 250, or 100 bp) with stop codon was constructed (Fig. 3a). Both donor and CRISPR/Cas9 vectors were co-introduced into the wild-type protoplasts, and the efficacies of the SDH disruption were evaluated by the depleting melanin deposition. White colonies were obtained with high efficiencies (10–25.58%) by co-introduction with pCRISPR/Cas-U6-1 site2 and each donor vector comprising 250–1000 bp homologous sequences (Fig. 3b). The pCRISPR/Cas9-U6-2 could produce more white colonies than pCRISPR/Cas9-U6-1, and it generated the mutant using only 100-bp homologous sequence (Fig. 3b). These results indicated that a single crossover-mediated gene disruption can be performed by using the shortened homology arms in P. oryzae.

Figure 3 SDH disruption using short homology sequences with CRISPR/Cas9. (a) Schematic representation of the donor vector having a short homology sequence. (b) The efficiencies of SDH disruption using the donor vector having a short homology sequence. Total colony: number of hygromycin B-resistant colonies obtained from triplicate experiments. White colony: number of hygromycin B-resistant colonies presenting the white phenotype. Efficiency: percentage of total white colonies. Full size image

Single crossover-mediated reporter gene knock-in with CRISPR/Cas9 system

To investigate whether the single crossover type HR can be applied for the reporter gene knock-in, we constructed a donor vector comprising a GFP fused SDH homologous sequence. In addition, we introduced silent mutations at the CRISPR/Cas9 target site to avoid the cleavage of the donor vector. Precisely, a single crossover recombination would introduce GFP at the C-terminus of the SDH gene (Fig. 4a). The hygromycin B resistant colonies with the GFP fluorescence were not obtained via introduction with the only donor vector, whereas co-introduction with the CRISPR/Cas9 and donor vectors helped in obtaining the transformants with the GFP fluorescence (Fig. 4b–d). Randomly selected nine fluorescent colonies comprised the desired silent mutations at the CRISPR/Cas9 target site of the SDH gene (Fig. 4e). These results indicated that the CRISPR/Cas9-induced single crossover-type recombination can be applied for the one-step reporter gene knock-in at the targeted genomic locus.

Figure 4 Single crossover-mediated reporter gene knock-in at the SDH locus with CRISPR/Cas9. (a) Schematic representation of the single crossover-mediated GFP knock-in at the SDH locus. The start and stop codons were deleted from SDH gene and the silent mutations were introduced at the CRISPR/Cas9 target site of this gene. GFP was fused to mutated SDH C-terminus. (b,c) GFP fluorescence in the wild type (b) and GFP-tagged transformant. (c) BF: Bright-field image, GFP: epifluoresence image. The bars are 500 µm. (d) The efficiencies of GFP knocked-in transformant. Total colony: number of hygromycin B-resistant colonies obtained from repeated experiments. Fluorescent colony: number of hygromycin B-resistant colonies presenting the GFP fluorescence. Efficiency: percentage of total GFP fluorescent colonies. (e) Sequences of the CRISPR/Cas9 target region in the wild type, donor vector, and fluorescent colonies of the transformants. Full size image

Functional and expression analysis of Spo11 using single crossover-mediated gene disruption and reporter gene knock-in

To assess whether the single crossover-mediated genome editing is effective for other genomic loci, we analyzed the functions and expression of SPO11 (MGG_10666) in P. oryzae. In Saccharomyces cerevisiae, Spo11 catalyzes DSB formation to act via topoisomerase-like reaction in meiosis24. Since asexual reproduction is predominantly observed in P. oryzae, the function of genes related to the meiotic recombination in asexual life cycle has not been characterized. We created two donor vectors for the functional disruption (Fig. S1) and GFP tagging of Spo11 (Fig. S2). When seven independent well-grown colonies obtained via co-introduction with CRISPR/Cas9 and donor vectors for functional disruption (pMK-PSPO) were subjected to sequencing analysis, three out of seven transformants had desired stop codons at the SPO11 gene (ca. 43%); however, the three mutants indicated no significant differences in the growth, conidiation, germination, and appressorium formation rates, compared with those of the wild-type strain. For the Spo11-GFP plus CRISPR/Cas9 co-transformation, ten hygromycin B-resistant colonies were randomly selected and two (20%) contained the desired allelic replacement based on PCR and sequencing analysis; however, the GFP fluorescence was not observed during the vegetative growth and appressorium formation. These results suggest that the single-crossover strategy could be applied universally across genomic loci. Furthermore, we have employed this strategy to functionally demonstrate that Spo11 does not play a role in the asexual life cycle of P. oryzae.