The CRISPR-Cas9 genome editing technology has recently been adapted for several species of filamentous fungi, including various Aspergilli species, Trichoderma reesei, Neurospora crassa, and Pyricularia oryzae among others. CRISPR-Cas9 induces specific DNA double strand breaks (DSBs) in the genome using a small specific RNA molecule as a guide. These breaks can then be used to destroy selected genes by relying on error-prone DNA repair by the non-homologous end-joining (NHEJ) to introduce mutations, or by increasing the efficiency of gene targeting in NHEJ proficient strains. Although elimination of a gene is an efficient tool towards understanding the function of the encoded protein, it is often advantageous to introduce small specific mutations to dissect the functionality of the protein in more detail. For example, it is possible to address the importance of individual amino-acid residues in protein function by changing single codons in the gene. Similarly, by introducing subtle changes in a multi-domain protein it is possible to understand the contribution of individual domains in the overall function of this protein. In applied sciences, site directed mutagenesis can be used to optimize enzyme function by protein engineering. Here we demonstrate a simple strategy for the generation of seamless point mutations, using short synthetic single stranded oligonucleotides and a CRISPR-Cas9 system in Aspergillus nidulans, and explore the parameters for efficient gene targeting, using this type of gene targeting substrate. We show that even in fungi with a well-established genetic toolbox CRISPR-Cas9 can still be a valuable addition, opening up new genetic engineering strategies.
Full conference title:
The Thirteenth International Aspergillus Meeting, EUROSITES La Chapelle, PARIS, FRANCE
- Asperfest 13 (2016)