Establishment of a cytosine base-editing system in Trichoderma koningiopsis

In this study, the authors developed two cytosine base-editing systems in T. koningiopsis based on CRISPR/nCas9 (D10A) by fusing either the conventional rAPOBEC1 or the newly engineered mini-SDD7 deaminase, and further optimized codon usage and genetic elements for this fungus. Both editing systems facilitated efficient C→T conversions in the genome without compromising strain growth. Notably, the mini-SDD7-CBE system achieved an editing efficiency of up to approximately 80% at the yak1 locus, significantly surpassing the performance of the rAPOBEC1-CBE system. Sequencing of multiple target sites, including ura5, yak1, ace1, and pk1, indicated that mini-SDD7-CBE exhibits a primary editing window approximately 16–20 base pairs upstream of the PAM, with additional flexibility within the 10–20 base pairs region. Cas-OFFinder predictions, in conjunction with TIDE analysis of 25 potential off-target sites, revealed no detectable off-target events, and the overall frequency of indels remained at the background levels.

To assess the multiplex editing capacity of mini-SDD7-CBE, the authors concatenated multiple sgRNA expression cassettes. Focusing on three key regulators of cellulase and hemicellulase production—yak1, ace1, and pk1—they constructed dual-target (yak1 and ace1) and triple-target (yak1, ace1, and pk1) editing vectors. A single transformation resulted in double knock-out mutants with an overall efficiency of approximately 10% and triple knock-out mutants with an efficiency of approximately 13%. Further analysis indicated that the overall success rate of multiplex editing was primarily constrained by the least efficient sgRNA, rather than by any intrinsic loss of efficiency with increasing target number. These findings show that mini-SDD7-CBE is fully capable of enabling one-step multiplex precise editing in T. koningiopsis, providing a practical strategy for constructing complex mutant combinations and performing systematic functional dissection.

Under cellulase-inducing fermentation conditions, the enzyme activities and transcriptional profiles of the resulting mutants were further evaluated. Ultimately, the authors obtained a high-cellulase-producing strain Δyak1 and a high-xylanase-producing strain Δyak1Δace1Δpk1, offering valuable references for the breeding of “customized” industrial enzyme-producing strains tailored to different application scenarios.