A High-Efficiency CRISPR–Cas9 Ribonucleoprotein Genome Editing System in Aspergillus fijiensis Enabled by Microhomology-Mediated End Joining

TL;DR

This paper introduces an efficient CRISPR–Cas9 system for editing the genome of Aspergillus fijiensis, a fungus important for industry, by leveraging microhomology-mediated repair.

Contribution

The study introduces a high-efficiency CRISPR–Cas9 RNP system for A. fijiensis using microhomology-mediated end joining for precise genome editing.

Findings

Using 5 bp microhomology arms in donor templates shifted repair from NHEJ to MMEJ, enabling precise edits in 92% of transformants.

A protoplast regeneration protocol with osmotic stabilization improved transformation efficiency in A. fijiensis.

RNP delivery alone produced frequent indels, but MMEJ donors enabled targeted insertions and deletions.

Abstract

Aspergillus fijiensis is an industrially important filamentous fungus, whose genetic analysis has been limited by the absence of species-specific tools. This study establishes an optimized CRISPR–Cas9 genome editing platform for A. fijiensis, from protoplast preparation to DNA repair pathway engineering. Antibiotic screening first identified hygromycin B and 5-FOA (5-fluoroorotic acid) as effective positive and counter-selection markers. A high-efficiency protoplast regeneration protocol was developed depending on specific osmotic stabilization and mycelial competence. Evaluation of a plasmid-based CRISPR system revealed that while autonomous replication was feasible, gene editing was constrained by low efficiency and a predominant bias toward NHEJ (non-homologous end joining). We implemented a Cas9–sgRNA RNP (ribonucleoprotein) delivery approach, with RNP delivery alone producing frequent indels. However, targeted integration remained inefficient when using conventional MMEJ (Microhomology-mediated end joining) donors. By employing donors containing short (5 bp) microhomology arms between cleavage sites, we effectively engaged the MMEJ pathway, enabling precise insertions and large-fragment deletions in 92% of the analyzed transformants. Donor templates containing minimal 5 bp microhomology sequences could effectively shift the predominant repair pathway from NHEJ to MMEJ. These findings demonstrate that MMEJ is the superior pathway with a unique mechanism for genome engineering in A. fijiensis, providing a versatile toolkit for unlocking the biotechnological potential of this recalcitrant species and a successful paradigm for establishing genetic systems in other species.

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