Creating Loss-of-Function Mutants of Neurospora crassa Using a Novel CRISPR/Cas9 System

Since its introduction, the CRISPR/Cas9 system has been used in many organisms for precise and rapid genome editing, as well as for editing multiple genes at once. This targeted mutagenesis makes it easy to analyze the function of a gene of interest (goi). The standard method for genetic manipulation of the model organism Neurospora crassa has been homologous recombination. It is well established and widely used to create knock-out or overexpression mutants. The recently developed CRISPR/Cas9 system is an addition to the toolkit for genetically manipulating N. crassa. For this protocol, a strain stably expressing the Cas9 endonuclease is required. After designing the gRNA with the online tool CHOP-CHOP, a synthetic gRNA is used to transform macroconidia via electroporation. Combining the goi-gRNA with a gRNA targeting the csr-1 gene as a selection marker allows for easy identification of colonies with mutations at the target site of the goi, since the obtained resistance to Cyclosporin A (CsA) allows for selecting editing events. The mutation type can be detected by PCR of the edited gene region followed by Sanger sequencing. This system is fast and easy to handle, offering an attractive alternative to homologous recombination, especially for targeting multiple genes simultaneously. Key features • This protocol allows the use of CRISPR/Cas9 in Neurospora crassa to create loss-of-function mutants. • It can be used to create loss-of-function mutants of multiple genes in one round of transformation. • Time-saving mutagenesis without the need for vector construction. • In combination with csr-1 as a selection marker, the screening for successfully edited genes of interest is reduced.