Fig. 1

The shematic diagram of the Arg-tRNA driven gRNA-Cas9/kov21 gene replacement system in P. oryzae. a The tandem Arg-tRNA driven gRNA expression cassette, which includes an 84 bp tRNA^Arg(ACG) gene of P. oryzae (red), gRNA spacer insertion region for inserting target sequence via BsmBI sites (green and underlined), gRNA scaffold (blue), and terminator (purple). b The strategy of the Arg-tRNA driven CRISPR/Cas9 system for efficient gene disruption in P. oryzae. The constructed Mo_tRNA^Arg-gRNA-Cas9 vector with 20-bp target sgRNA co-transformed with donor pKOV21 vector into protoplast to generate sgRNA and induce gene replacement. Two pairs of primers, Check up/Hyg up and Check down/Hyg down are used to confirm correct gene knock-out mutants