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SPECIAL SESSION: Current Trends and Directions in CRISPR/Cas9 Technology for Filamentous Fungi and Oomycetes

Versatile applications of CRISPR/Cas9 mediated genome editing in fungal and oomycete pathogens
Francis Fang - Duke University. Ianiri Giuseppe- Duke University, Bhagya Thimmappa- Molecular Biology and Genetics Unit, JNCASR, Marco Coelho- Duke University, Klaas Schotanus- Duke University, Yadav Vikas- Duke University, Joseph Heitman- Duke University, Minou Nowrousian- Lehrstuhl fu

CRISPR/Cas9 systems have been shown to be a useful tool for modifying the genomes of various fungal and oomycete pathogens. Previously, we developed an oomycete-optimized CRISPR/Cas9 system which has been used for efficient targeted mutagenesis and gene replacement in the model oomycete pathogen Phytophthora sojae. Here, we show several additional cases in which a simplified and generalized “all-in-one” CRISPR/Cas9 expression plasmid has been efficiently used for generating mutants of different targets and purposes in P. sojae. We constructed a GFP-tagged endogenous CENP-A (a kinetochore protein involved in cell division) allele by gene replacement, and conducted ChIP-seq to define the nature of centromeres in oomycetes. All the identified P. sojae centromeric regions are rich in transposons and show low transcription. In addition, we developed a dual CRISPR/Cas9 method in the basidiomycetous fungus Cryptococcus neoformans. This approach was successfully used for simultaneous genome editing of two difficult targets and for allelic exchanges. We generated a double mutant lacking the Rad51 and Rad52 proteins that are required for homologous recombination, which would not be possible to isolate by conventional sequential single gene deletion. Rad51 and Rad52 are required for quelling in Neurospora crassa, in contrast, we found they are not required for RNAi-mediated mitotic-induced silencing (MIS) in C. neoformans. Using the dual CRISPR system, we also tested by allelic exchange the functions of the essential ribosomal genes RPL22a and RPL22? encoded by the mating-type locus.