Overcoming diverse homologous recombinations and single chimeric guide RNA competitive inhibition enhances Cas9‐based cyclical multiple genes coediting in filamentous fungi

Deciphering the complex cellular behaviours and advancing the biotechnology applications of filamentous fungi increase the requirement for genetically manipulating a large number of target genes. The current strategies cannot cyclically coedit multiple genes simultaneously. In this study, we firstly revealed the existence of diverse homologous recombination (HR) types in marker-free editing of filamentous fungi, and then, demonstrated that sgRNA efficiency-mediated competitive inhibition resulted in the low integration of multiple genetic sites during coediting, which are the two major obstacles to limit the efficiency of cyclically coediting of multiple genes. To overcome these obstacles, we developed a biased cutting strategy by Cas9 to greatly enhance the desired HR type and applied a new selection marker labelling strategy for multiple donor DNAs, in which only the donor DNA with the lowest sgRNA efficiency was labelled. Combined with these strategies, we successfully developed a convenient method for cyclically coediting multiple genes in different filamentous fungi. In addition, diverse HRs resulted in a useful and convenient one-step approach for gene functional study combining both gene disruption and complementation. This research provided both a useful one-step approach for gene functional study and an efficient strategy for cyclically coediting multiple genes in filamentous fungi.