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POSTERS: Biotechnology and genetic engineering

CRISPR/Cas9-enabled multiplex genome editing and locus-dependent DNA repair in Magnaporthe oryzae
Matthew Wheatley - The Pennsylvania State University. Yinong Yang- The Pennsylvania State University

Magnaporthe oryzaeis the causal agent of rice and wheat blast diseases and poses a major threat to rice and wheat production worldwide. To facilitate functional discovery of the fungal genes and subsequent elucidation of its virulence mechanisms, it is important to develop and improve CRISPR/Cas9 tools and methods for genome editing and targeted mutagenesis in M. oryzae. In this study, we successfully performed CRISPR/Cas9-mediated multiplex genome editing in the rice blast fungus using the polycistronic tRNA-gRNA (PTG) strategy. Upon creation of double stranded breaks (DSBs) by Cas9, targeted gene mutation in M. oryzaewas achieved by either non-homologous end joining (NHEJ) or homology-directed repair (HDR) depending on the targeted loci. In absence of donor templates, Cas9-induced DSBs frequently triggered genomic rearrangement, leading to the loss of PCR-based amplification. By providing donor templates, however, HDR-mediated precise genome editing was achieved at the efficiency up to 100% in PCR positive transformants when targeting a single locus. The PTG-based multiplex genome editing via HDR also successfully led to the generation of double and triple gene mutants. The resulting CRISPR/Cas9 tools and strategies from this study are expected to aid in the efficient genome editing and functional genomics analysis for M. oryzaeand other fungal species.