Development of an Efficient C-to-T Base-Editing System and Its Application to Cellulase Transcription Factor Precise Engineering in Thermophilic Fungus Myceliophthora thermophila

Myceliophthora thermophila is a thermophilic fungus with great potential in biorefineries and biotechnology. The base editor is an upgraded version of the clustered regularly interspaced short palindromic repeats (CRISPR)-dependent genome-editing tool that introduces precise point mutations without causing DNA double-strand breaks (DSBs) and has been used in various organisms but rarely in filamentous fungi, especially thermophilic filamentous fungi. Here, for the first time, we constructed three cytosine base editors (CBEs) in M. thermophila, namely, evolved apolipoprotein B mRNA-editing enzyme catalytic subunit 1 (APOBEC1) cytosine base editor 4 max (Mtevo-BE4max), bacteriophage Mu Gam protein cytosine base editor 4 max (MtGAM-BE4max), and evolved CDA1 deaminase cytosine base editor (Mtevo-CDA1), and efficiently inactivated genes by precisely converting three codons (CAA, CAG, and CGA) into stop codons without DSB formation. The Mtevo-CDA1 editor with up to 92.6% editing efficiency is a more suitable tool for cytosine base editing in thermophilic fungi. To investigate the function of each motif of the cellulase transcription factor M. thermophila CLR-2 (MtCLR-2), we used the Mtevo-CDA1 editor. The fungal-specific motif of MtCLR-2 was found to be strongly involved in cellulase secretion, conidium formation, hyphal branching, and colony formation. Mutation of the fungus-specific motif caused significant defects in these characteristics. Thus, we developed an efficient thermophilic fungus-compatible base-editing system that could also be used for genetic engineering in other relevant filamentous fungi. IMPORTANCE A CRISPR/Cas-based base-editing approach has been developed to introduce point mutations without inducing double-strand breaks (DSBs) and attracted substantial academic and industrial interest. Our study developed the deaminase-cytosine base-editing system to efficiently edit three target genes, amdS, cre-1, and the essential cellulase regulator gene Mtclr-2, in Myceliophthora thermophila. A variety of point mutations in the target loci of the DNA-binding domain and fungus-specific motif of M. thermophila CLR-2 (MtCLR-2) were successfully generated via our base editor Mtevo-CDA1 to elucidate its function. Here, we show that the DNA-binding domain of MtCLR-2 is important for the fungal response to cellulose conditions, while its fungus-specific motif is involved in fungal growth. These findings indicate that our base editor can be an effective tool for elucidating the functions of motifs of target genes in filamentous fungi and for metabolic engineering in the field of synthetic biology.