# 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.

## Key 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.