Expanding the Genetic Toolkit: Adenine and Cytosine Base Editors for Efficient Gene Disruption in Aspergillus Niger

Despite revolutionizing fungal genetic engineering, conventional CRISPR/Cas9-mediated knockouts rely on DNA double-strand breaks (DSBs), which can cause unwanted insertions and deletions, chromosomal abnormalities, and cytotoxicity. Base editors such as adenine base editors (ABEs), which convert A•T to G•C, and cytosine base editors (CBEs), which convert C•G to T•A, offer a safer alternative by enabling precise single-nucleotide changes without introducing DSBs. To overcome the limitations of traditional genome editing in filamentous fungi, we developed efficient base-editing systems in Aspergillus niger . For the first time, we constructed an ABE in A. niger , achieving up to 80% editing efficiency and inducing precise A-to-G mutations at conserved intron sites that disrupted gene function through mRNA mis-splicing. We also developed a highly efficient CBE system, capable of introducing premature stop codons with 50–100% efficiency. Furthermore, we established gene disruption approaches by targeting start codons: ABE-mediated A-to-G conversions (ATG-to-ACG and ATG-to-GTG) and CBE-mediated C-to-T conversion (ATG-to-ATA). To broaden the editing scope, we implemented a Cas9-NG variant recognizing a relaxed PAM sequence requiring only a single guanine (G), enabling editing at start codons and splice sites. Additionally, our base-editing systems enable multiplex gRNA delivery and marker-free editing of multiple genes. Together these improvements increase the number of genes targetable for disruption by base-editing in A. niger by 26.3% and enable near-complete coverage of 96% of the coding genes. Overall, this work demonstrates the potential of ABE and CBE systems as versatile, efficient, and safer alternatives to DSBs-based gene disruption in filamentous fungi.