Seamless gene editing in Aspergillus species, using CRISPR-Cas9

Many fungi are both excellent degraders of biomass and natural producers of industrially interesting compounds, making them good candidates for cell factories. Several members of the genus Aspergillus are successfully used as industrial cell factories for production of organic acids, enzymes and other primary or secondary metabolites, and many other Aspergilli are currently being sequenced and might possess traits making them similar suitable as potential cell factories. Yields from such cell factories can be greatly enhanced by employing genetic engineering strategies, however there are several obstacles slowing down the process.

The harnessing of the prokaryotic and archaeal immune mechanism CRISPR (clustered regularly interspaced short palindromic repeats) as a tool for genetic engineering in eukaryotes, has proved to be a powerful technology. CRISPR/Cas9 introduces specific DNA double strand breaks (DSB) with high precision, which in turn can be employed to efficiently stimulate gene targeting. Consisting of two components, an RNA guided nuclease Cas9 and a chimeric guide RNA (gRNA), a specific DSB can be produced in the host organism, which can be utilized to facilitate precise gene editing. The cleavage target site is determined by 20 base pairs (bp) in the gRNA, and by exchanging those 20 bp, Cas9 can be programmed to target a specific chromosomal location with few constraints. The technology has had a huge impact on genetic engineering of organisms, such as plants or mammalian cells where gene targeting is notoriously inefficient, but has only recently been adapted to filamentous fungi.

When using conventional strategies for genetic engineering in filamentous fungi, most strategies results in a genetic selection marker being left behind at the site of the edit, which can affect metabolism and negatively impact downstream processing. Here we present methods allowing for seamlessly inserting or deleting genes, for precisely introducing point mutations without changing the surrounding sequence, and a simple assay to easily identify efficient gRNAs. Together these methods provide a valuable addition to the genetic toolbox of several species of industrial relevant Aspergillus species, which can greatly accelerate the development of new fungal cell factories.