Fig. 2

Deletion of the cre1 gene in T. reesei by the tRNA−gRNA array-based multi-sgRNA processing platform. A Schematic diagram of the cre1 gene deletion process in T. reesei. After transformed to the parental strain QM53, two sgRNAs (sgRNA-cre1U and sgRNA-cre1D) were co-expressed from the tRNA−sgRNA array cassette named as 5StsgRNA (cre1*2). Subsequently, the Cas9 endonucleases were recruited to the target sites of cre1 by sgRNA-cre1U and sgRNA-cre1D to generate two double-strand breaks (DSBs, ▲ represents the sites for DSB) adjacent to the PAM sequence. After losing of the middle fragment of the two DSBs, the chromosome could be repaired by non-homologous end-joining (NHEJ) to complete deletion of cre1. The final Δcre1 strain was named as QMC1. B Growth of the Δcre1 strain QMC1 and the parental strain QM53 on the MM plate containing Avicel (0.5%) as the sole carbon source, the MM plate containing Avicel (0.5%) plus glucose (1.0%) as the mixed carbon sources and the PDA plate. C Sequence alignment of the cre1 target locus in QMC1. The sequencing result of cre1 from QMC1 was shown and the cre1 sequence from the parental strain QM53 was listed as the wild-type reference gene (the two PAM sequences are highlighted in yellow and protospacer sequences in pink)