Title: 7th Slovenian Symposium on Plant Biology with International Participation
Abstract: The challenges that modern agriculture faces to sustain food production are many and varied.One of the most important of these is the stabilisation of crop yield through the development of superior germplasm.The coevolution of plants and pathogens has resulted in a multifaceted, ongoing arms-race between the two, which seems to be slowly tipping towards the pathogens, especially considering the increasing numbers of infectious plant diseases caused by fungal, bacterial and viral pathogens.This situation necessitates the implementation of cutting edge technologies, like genome editing, for plant disease resistance breeding.Genome editing comprises site-directed mutagenesis by the introduction of targeted double-strand breaks in DNA through the action of programmable nucleases, and the subsequent fixing of these breaks by one of two natural DNA repair mechanisms.CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/(CRISPR-associated protein9), the most widely used of these technologies, allows precise, targeted editing of commercial crop varieties, thus offering a viable, and much faster, alternative to traditional breeding methods.From a plant pathology perspective, additional advantages of this approach are that either key components of the host immunity system (e.g.susceptibility or resistance genes), or genome regions of the pathogen(s) (e.g.essential genes for replication) can be targeted, and that these modifications can be made in a non-transgenic manner.CRISPR/Cas9 technology is based on an age-old adaptive immune system found in bacteria and archaea, aimed at protecting these organisms against virus infections.Scientific intervention has seen this system being adapted and optimised in a number of ways to allow for pin-point precision editing of the genome target sequences, potentially resulting in either knock-out or knock-in mutations.Here, we discuss the application of CRISPR/Cas9 technology in plant pathology.Aspects that are covered include experimental strategies for sgRNA design, choice of nuclease, vector construction, delivery systems, detection and analysis of in vivo mutagenesis, and analysis of off-target effects.Examples of CRISPR/Cas9 approaches to introduce resistance against fungal, bacterial and viral pathogens will be discussed and, finally, two case studies of current CRISPR/Cas9 research projects (grapevine and soybean) in our laboratory will be highlighted.