Cyclosporin A-resistance based gene placement system for Neurospora crassa

DNA introduced into Neurospora crassa are usually inserted at random ectopic sites of the genome, often in multiple copies. To facilitate the study of gene expression and function, transformation by a single-copy of a gene at a defined locus is desired. Although several targeted gene placement methods are available for N. crassa, they all require a specific genetic background in the recipient. We describe here the development of a new locus for targeted gene placement that does not require any pre-existing marker in the target strain. Our system takes advantage of the fact that disruption of the csr-1 gene, which encodes the cyclosporin A-binding protein, leads to the resistance to cyclosporin A. By cloning a gene of interest into a csr-1 knock-in vector and transforming a fungus with it, one can easily insert any gene, in single-copy, into a defined locus.