CRISPR/Cas9 nickase-mediated cytosine base editing in Trichoderma koningiopsis.

Base editors are regarded as reliable and precise editing tools due to their ability to efficiently and accurately modify the target genes without inducing double-strand breaks (DSBs). Notably, base editors offer distinct advantages in base editing, particularly in multiplex gene editing. However, application of base editors in the filamentous fungus Trichoderma remains underdeveloped. In this study, two cytosine base editors (CBE), designated as rAPOBEC1-CBE and mini-SDD7-CBE, respectively, were constructed in Trichoderma koningiopsis. Three targets related with cellulase production were chosen for testing editing efficiency of these two systems. The mini-SDD7-CBE system exhibited a maximum editing efficiency of 80%, with the editing window predominantly situated at positions 16 to 20 upstream of the PAM sequence in T. koningiopsis. Utilizing the mini-SDD7-CBE system, simultaneous editing of two and three genes was successfully executed. The overall editing efficiency was primarily influenced by the lowest editing efficiency of an individual single guide RNA (sgRNA). This study provides a convenient tool for the precise genome engineering of Trichoderma, thereby facilitating the extensive application of base editing techniques in filamentous fungi.