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

Abstract

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 .

MtTRC-1, a Novel Transcription Factor, Regulates Cellulase Production via Directly Modulating the Genes Expression of the Mthac-1 and Mtcbh-1 in Myceliophthora thermophila

“…In ascomycetous filamentous fungi, the expression of (hemi-)cellulase genes is commonly directly regulated by various transcription factors ( 18 , 21 – 24 ). However, to date, only a few transcription factors, including MtXlr-1 ( 13 , 43 ), MtCRE-1 ( 44 ), and MtCLR-4 ( 45 ), and MtCLR-2 ( 46 ), have been confirmed to participate in the direct transcriptional regulation of (hemi-)cellulase genes in M. thermophila . Our transcriptome data reflected the significantly downregulated expression of cellulase genes in the Δ Mttrc-1 mutant grown on Avicel.…”

“…We previously characterized the new conserved Zn2Cys6 transcription factors NcCLR-4 and MtCLR-4 in N. crassa and M. thermophila and demonstrate that NcCLR-4/MtCLR-4 is a pivotal regulator for cellulolytic gene expression by directly regulating the expression of the essential transcriptional activators CLR-1 or CLR-2 and the key component of the cAMP signaling pathway adenylyl cyclase cr-1 gene ( 45 ). Recently, we investigated the gene functions of the CLR-2 ortholog of MtCLR-2 in M. thermophila using the base editor and classical CRISPR/Cas9 systems ( 46 ). Our results show that the DNA-binding domain of MtCLR-2 is important for the fungal response to cellulose conditions and its fungus-specific motif is involved in fungal growth ( 46 ).…”

“…Recently, we investigated the gene functions of the CLR-2 ortholog of MtCLR-2 in M. thermophila using the base editor and classical CRISPR/Cas9 systems ( 46 ). Our results show that the DNA-binding domain of MtCLR-2 is important for the fungal response to cellulose conditions and its fungus-specific motif is involved in fungal growth ( 46 ).…”

The expanding CRISPR toolbox for natural product discovery and engineering in filamentous fungi

“…46,47 In lamentous fungi dCas9 cytosine base editors, which convert cytidine (C) to thymine (T), have been used in Aspergillus niger and Myceliophthora thermophila (see Section 3.3). 48,49 CRISPR/Cas-based tools have also been built for transcriptional modulation. These tools are generally built with dCas proteins, including dCas9 and dCas12a, which can be programmed to deliver effector proteins to specic regulatory regions (Fig.…”

Use of CRISPR‐Cas tools to engineer Trichoderma species

“…However, due to more stringent requirements of PAM sequences, it will reduce the number of practicable target sites (Kaya et al, 2016 ). The recent development of prime editing and base editor provides more potential strategies for engineering Trichoderma genome (Anzalone et al, 2019 ; Zhang et al, 2022 ).…”