A CRISPR/Cas9 based toolbox for efficient genome editing of filamentous fungi

Abstract

Large numbers of fungi are currently being fully sequenced and will contribute dramatically to our understanding of fungal biology. However, the fact that gene targeting is inefficient in most fungal species hampers exploitation of the genome sequences. This problem has been significantly reduced after efficient CRISPR/Cas9 gene editing has been introduced in several different fungal species. A challenge of the CRISPR/Cas9 system is to deliver gRNAs to form the Cas9 ribonuclease. Several approaches have been presented in the literature including methods where gRNAs are co-transformed into the cells along with the gene editing DNA substrates, methods where the gRNA is produced by RNA polymerase III, and methods where the gRNA is liberated by ribozymes from a larger transcript produced by RNApolymerase II. Since the different methods have different advantages/disadvantages, we envision that they may work with different efficiencies in different fungal species. We have therefore developed a flexible CRISPR/Cas9 toolbox adapted for filamentous fungi to facilitate genome editing. Our toolbox includes bio-bricks containing e.g. different genetic markers and polymerase promoter types allowing for a rapid and efficient vector assembly and bricks that allow for quick insertion of new genes into strong expression sites for heterologous expression. Moreover, it includes bricks to facilitate trouble shooting including a cas9-RFP reporter gene to evaluate Cas9 levels in new hosts and a system allowing the efficiency of individual gRNA species to be tested in vivo. Using our toolbox, we have successfully edited the genomes of more than 10 species and used it to make a different range of genetic alterations including site specific mutations by using oligonucleotides as repair templates and deletions. In this way we have linked secondary metabolites to genes in species that have not previously been genetically engineered. For strains where we plan to do extensive gene targeting, we typically use CRISPR to mutate thepyrG gene, hence, producing a marker than can be selected/counter selected. Next, we mutate a gene in the NHEJ pathway to produce a strain where gene targeting is very efficient. We will show how this strategy can be used investigate the biosynthetic pathway of gene clusters. Lastly, we will show how markers and mutations in NHEJ genes can be easily reverted to wild-type if a wild-type background is desirable in the subsequent analyses