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

Abstract

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.

Exploring the multi-level regulation of lignocellulases in the filamentous fungus Trichoderma guizhouense NJAU4742 from an omics perspective

“…Gene editing was performed using hygromycin B (HygB) for screening according to our previous method [ 22 ]. Briefly, the donor DNAs of Tgxyr1 , Tgclr-2 , Tgace1 and Tgace2 were constructed in the form of UF (upstream fragment of the target gene) + hph -EC ( hph gene expression cassette) + DF (downstream fragment of the target gene) by fusion PCR using CloneAmp HiFi PCR Premix (TAKARA, Japan).…”

“…Protoplasts of strain NJAU4742 were then prepared using 7.5 mg·mL − 1 lysing enzyme (Sigma: L1412, China) dissolved in solution A (1.2 M sorbitol, 0.1 M KH 2 PO 4 , pH 5.6). Next, 10 µl DNA (≥ 1 µg) was transformed into 200 µL protoplasts (≥ 1 × 10 8 protoplasts/mL) by the PEG-CaCl 2 method [ 22 ]. Protoplast regeneration was achieved by spreading the transformation mixture evenly onto 1 M sucrose-containing PDA plates for 24 h at 28 ℃, which were then covered with 0.2 mg·mL − 1 HygB-containing PDA medium.…”

“…The Trichoderma guizhouense NJAU4742, an efficient lignocellulose degrader and plant beneficial fungus [ 20 – 22 ], was mass-produced by the solid fermentation mainly using crop straws as the carbon source in biofertilizer area of China. Thus, in this study, strain NJAU4742 was used to investigate the regulation of PBDEs when induced by different polysaccharides or under carbon starvation.…”

Intracellular kynurenine promotes acetaldehyde accumulation, further inducing the apoptosis in soil beneficial fungi Trichoderma guizhouenseNJAU4742 under acid stress

“…After 36 h, the spontaneous homologous strains germinated and were transferred to MM medium containing 5-fluoroorotic acid and urea. The transformants similar with WT were selected for the subsequent study, and the spontaneous homologous recombination mutation was verified by PCR reaction (Miao et al, 2021).…”

Simultaneous manipulation of multiple genes within a same regulatory stage for iterative evolution of Trichoderma reesei

“…The past decades have witnessed the successes in genome sequencing of many filamentous fungi as well as in introducing multiple genes into a single filamentous fungal cell such as T. reesei [ 9 ], Trichoderma guizhouense [ 10 ], and M. thermophila [ 11 ]. The availability of sequenced genomes suggests that uncharacterized homologs of known regulatory genes in the prevalent fungi might play similar, orthologous roles in non-canonical filamentous fungi.…”