Introducing multiple-gene mutations in Pleurotus ostreatus using a polycistronic tRNA and CRISPR guide RNA strategy

Abstract

The white-rot fungus Pleurotus ostreatus is an agaricomycete that is frequently used in molecular genetics studies as many useful tools are applicable to the fungus. In particular, efficient gene targeting using homologous recombination and CRISPR/Cas9 enables the introduction of a mutation in the gene of interest for functional analysis. Multiple genes encoding various lignocellulose-degrading enzymes are predicted to be present in the genome; therefore, analyses of multiple-gene mutants are required to elucidate the mechanisms underlying lignocellulose degradation by P. ostreatus. Conventional tools for generating multiple-gene mutations in P. ostreatus are laborious and time-consuming. Therefore, more efficient and practical methods are needed. In this study, we introduced CRISPR/Cas9-assisted multiple-gene mutations using a polycistronic tRNA and CRISPR guide RNA approach. The frequency (triple-gene mutation in fcy1, vp2, and 62 347) was only 3.3% when a tetracistronic tRNA-sgRNA containing four different sgRNAs targeting fcy1, vp2, vp3, or 62 347 was expressed. It increased to 20% (triple-gene mutation in vp1, vp2, and vp3) after a tricistronic tRNA-sgRNA was expressed with replaced/modulated promoter and tRNA sequences. This study demonstrated, for the first time, the applicability of a strategy to induce multiple-gene mutations in P. ostreatus in a transformation experiment.

Experimental evidence that lignin‐modifying enzymes are essential for degrading plant cell wall lignin byPleurotus ostreatususingCRISPR/Cas9

“…The resulting plasmids were named pCcPef3‐126‐ vp2 , pCcPef3‐126‐ vp3 , pCcPef3‐126‐ mnp3 , and pCcPef3‐126‐ mnp6 , respectively. It was confirmed that a mutation in each mnp / vp was frequently (75%–100%) introduced into the hygromycin B‐resistant strains transformed with each plasmid (Xu et al, 2022; data not shown).…”

“…The sgRNA insertion into the plasmids was verified using Sanger sequencing. The resulting plasmids were named respectively. It was confirmed that a mutation in each mnp/vp was frequently (75%-100%) introduced into the hygromycin B-resistant strains transformed with each plasmid (Xu et al, 2022;data not shown).…”

“…The different sgRNA sequences used to target vp3 , mnp2 , mnp3 , mnp6 , and lac2 were designed as described by Boontawon, Nakazawa, Horii, et al (2021) and Boontawon, Nakazawa, Inoue, et al (2021). The targeting vp2 was designated by Xu et al (2022).…”

“…To construct the pCcPef3-126-based plasmids containing two expression cassettes of two different sgRNAs, inverse PCR was conducted using pCcPef3-126-mnp3 (A) and pCcPef3-126-mnp6 (B) with IY2/IY3, separately. The sgRNA-expressing cassettes of vp2 (C) and vp3 (D) and the P. ostreatus u6a promoter (Xu et al, 2022;Scaffold_9:233,432-233,925) (E) were also amplified from pCcPef3-126-vp2, pCcPef3-126-vp3, and the PC9 genome with IY6/IY5, IY7/IY5, and IY8/IY9, respectively. The three fragments, (A) (C) (E) or (B) (D) (E), were fused using the NEBuilder Hi-Fi DNA Assembly Cloning Kit (New England Biolabs, Massachusetts).…”

“…An efficient plasmid-based CRISPR/Cas9 was recently established in the fungus (Boontawon, Nakazawa, Horii, et al, 2021;Boontawon, Nakazawa, Inoue, et al, 2021). Using this technique, multiple-gene mutants can be obtained in one transformation experiment by expressing different sgRNAs concomitantly (Xu et al, 2022). In this study, we generated multiplegene mutants of mnp/vp/lac and examined/compared their wood lignin-degrading abilities to examine whether the LMEs play a dispensable role in wood lignin degradation by P. ostreatus.…”