Background

Filamentous fungi are a source of numerous pharmaceuticals and industrial enzymes, including penicillin. With the widespread use of genome sequencing, it has become clear that a large number of 'silent biosynthetic gene clusters (BGCs)' exist in the genomes of filamentous fungi, which are not currently expressed. The problem is that it is extremely difficult to experimentally activate this potential. Existing CRISPR-Cas9-based editing relies on double-strand breaks (DSBs), and due to the inherently low homologous recombination efficiency of filamentous fungi, it is difficult to achieve precise base substitutions or large-scale insertions and deletions.

Prime editing is a next-generation genome editing technology that directly inserts the desired sequence without DSBs, but applying existing systems optimized for mammalian cells directly to filamentous fungi results in a significant decrease in editing efficiency.

Key Findings

The research team developed a filamentous fungi-specific prime editor, fPE7max. This was achieved through multi-layered engineering tailored to the cellular environment of filamentous fungi, including codon optimization of the reverse transcriptase-Cas9 nickase fusion protein, rearrangement of the nuclear localization signal (NLS), and stabilization of the prime editing guide RNA (pegRNA) structure.

fPE7max can perform single-base substitutions, kilobase (kb)-scale insertions, and multi-kb deletions using a single platform. In particular, it has been shown to work in a variety of industrially important filamentous fungal species, such as Aspergillus and Penicillium, overcoming the previous limitation of having to construct species-specific tools separately.

The researchers demonstrated the power of this tool by precisely editing the translation-regulatory element of a pleiotropic metabolic regulator. By fine-tuning the regulatory sequence at the translational level, rather than at the transcriptional level, they selectively activated biosynthetic pathways that were previously inaccessible through conventional overexpression or knockout strategies. As a result, a new fungal-derived metabolite that has not been previously reported was discovered. By precisely manipulating a single regulatory factor, multiple downstream pathways were simultaneously activated.

Significance and Prospects

The silent BGCs in filamentous fungal genomes are estimated to be several times more numerous than the known active clusters. fPE7max provides a tool to systematically explore this vast chemical dark matter. The fact that editing is performed without DSBs means that genomic instability is low, and the ease with which pegRNA design principles validated in one species can be transferred to other filamentous fungal species is also a practical advantage.

However, prime editing efficiency can vary depending on the secondary structure of the target sequence and the cell cycle stage, so the accumulation of species- and locus-specific optimization data is necessary for it to become a universal pipeline. In the case of large-scale insertions, the trade-off between editing accuracy and insertion size also remains a challenge to be quantitatively elucidated in the future.