Developing safeguarding CRISPR-Cas9 gene drive in the plant pathogenic fungus, Sphaerulina musiva.

April 26, 2023 Dr. Joanna Tannous Poplars are an important species of tree and the pathogenic fungus, Sphaerulina musiva, is a significant problem where Poplars are cultivated. Dr. Tannous from Oak Ridge National Laboratory presents her ideas and recent efforts to develop genetic and genomics tools that enable the molecular genetic investigation of S. musiva as well as the development of gene drive genetic biocontrol strategies. 00:00 Speaker Introduction 01:16 Introductory Remarks 01:43 Outline of talk 02:23 Personal Background 03:43 Fungal Genetic Background and Experiences 05:07 The Fungal Kingdom: divers and essential roles in the environment 07:40 Background: The versatile poplar and its importance 08:25 Background: Sphaerulina musiva 09:33 Advancement in disease management 10:53 Overall approach 12:07 Development of a gene drive in S. musiva to mitigate Populus disease 13:46 Development of CRISPR transformation system in S. musiva 16:56 Identification of genetic targets for gene drive development 20:56 Transcriptomics analysis 24:53 Atpenin BGC genes missing or non-functional biosynthetic genes 25:48 Implementation of a safeguarding CRISPR-Cas9 gene drive in S. musiva to mitigate populus diseases. 28:06 Experimental approach – split gene drive Experimental overview 28:56 Hce2 guide RNA design 29:36 G236 strains across the US and Canada 30:49 Plasmid Map. 31:56 Experimental plan 32:30 Results – validated with pcr, sequencing 34:07 Second transformation – Cas construct. 35:04 Developing mating assay for validation of the gene drive. 35:58 Life cycle of S. musiva 38:20 Diagram of mating assay 40:16 Conclusions 41:19 Acknowledgements 41:54 Questions & Discussion

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