Fig. 1

Graphical overview of the tRNA−gRNA array-based CRISPR/Cas9 system for multiplex genome editing in T. reesei. The initial sgRNA vector is composed of the fragments containing the Cas9 cassette, the tRNA−gRNA array cassette, the PtrA cassette (selection marker) and AMA1 (autonomously replicating sequence). The tRNA−gRNA array cassette located on the initial sgRNA vector carries the 5S rRNA promoter, the tandemly arrayed tRNA−gRNA architecture (including tRNA, protospacer and sgRNA scaffold) and the T₆ terminator. Then, the cassette serves as a template for PCR amplification to generate new cassettes that can carry multiple sgRNAs (the detailed PCR amplification process is described in Materials and Methods). The multi-sgRNA cassette is ligated into the empty vector to generate the multi-sgRNA expression vector. After transformed to T. reesei, the Cas9 endonucleases and the primary transcript of the tRNA−gRNA array cassette are expressed. Subsequently, the primary transcript is cleaved by endogenous RNase P, RNase Z and exonuclease at the 5ʹ and 3ʹ-ends of tRNA to release multiple functional sgRNAs. The excised functional sgRNAs and the Cas9 endonucleases are then assembled into the mature Cas9/sgRNA complex in vivo, which can target multiple sites simultaneously for multiplex genome editing. In addition, the AMA1-based plasmids can be lost after successive subculture, enabling the recycling of selection markers and multiple rounds of genetic engineering