CRISPR/Cas9-mediated genome editing directed by a 5S rRNA–tRNAGly hybrid promoter in the thermophilic filamentous fungus Humicola insolens

Abstract

Background Humicola insolens is a filamentous fungus with high potential of producing neutral and heat- and alkali-resistant cellulase. However, the genetic engineering tools, particularly the genome-editing tool, are scarce, hindering the study of cellulase expression regulation in this organism. Results Herein, a CRISPR/Cas9 genome-editing system was established in H. insolens based on a hybrid 5S rRNA–tRNAGly promoter. This system is superior to the HDV (hepatitis delta virus) system in genome editing, allowing highly efficient single gene destruction in H. insolens with rates of deletion up to 84.1% (37/44). With this system, a putative pigment synthesis gene pks and the transcription factor xyr1 gene were disrupted with high efficiency. Moreover, the extracellular protein concentration and cellulase activity largely decreased when xyr1 was deleted, demonstrating for the first time that Xyr1 plays an important role in cellulase expression regulation. Conclusions The established CRISPR/Cas9 system is a powerful genetic operation tool for H. insolens, which will accelerate studies on the regulation mechanism of cellulase expression and engineering of H. insolens for higher cellulase production.

CRISPR/Cas9 mediated gene editing of transcription factor ACE1 for enhanced cellulase production in thermophilic fungus Rasamsonia emersonii

“…2B). Similarly, during CRISPR/Cas mediated gene editing of the thermophilic fungus H. insolens, Fan and coworkers [21] have also reported frame-shift mutations upstream of the PAM site involving either a single nucleotide deletion or insertion.…”

“…In another report, three cytosine base editors (CBEs; Mtevo-BE4max, MtGAM-BE4max, and Mtevo-CDA1) in M. thermophila were used for gene inactivation by precisely changing three codons (CAA, CAG, and CGA) into stop codons without the production of DSBs [20]. In H. insolens, a hybrid 5S rRNA-tRNA Gly promoter based CRISPR/Cas9 based disruption of pigment synthesis gene pks and the transcription factor xyr1 gene was developed [21], demonstrating the significance of CRISPR technology in genetic engineering of thermophilic fungi.…”

Editing Aspergillus terreus using the CRISPR-Cas9 system

“…(2013) demonstrated that CRISPR/Cas9-mediated genome editing could be applied to Saccharomyces cerevisiae ( 17 ), the approach was quickly introduced into a number of filamentous fungi including Trichoderma reesei ( 18 ), Neurospora crassa ( 19 ), a number of Aspergillus species ( 20 ) and Penicillium chrysogenum ( 21 ). Up to date, the CRISPR-Cas9 genome-editing methods have been established in >40 fungal species ( 12 , 22 , 23 ), and most of them were conducted using a single-guide ribonucleic acid (sgRNA) and Cas9 to introduce a DNA double-strand break into the target sequence and donor DNA with homology arms to introduce the desired genetic change ( 12 ). In contrast to other Aspergillus species, relatively few genetic manipulation methods have been established for A. terreus research.…”