Collaborative Research: TRTech-PGR TRACK: Discovery and characterization of small CRISPR systems for virus-based delivery of heritable editing in plants.

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Given current crop productivity projections, agricultural practices will be inadequate to meet future global food demands. Humanity’s ability to address this problem will largely depend on the efficiency with which novel genetic diversity can be created and introduced into plant breeding programs. New genome editing techniques allow for a more precise introduction of genetic diversity, but the current methods are very slow and inefficient and only work in some crop species. This project aims to discover novel genome editing systems that can be more widely and quickly deployed in a large variety of crop species, to enhance plant breeding to make crops with higher yields and resistance to drought, pests, and extreme temperatures. The new tools will also be useful for orphan crop species that do not receive sufficient attention from the plant biotechnology industry. The project will also include a structured approach to involving undergraduate student researchers from diverse backgrounds through different programs at the two participating University of California campuses, UCLA and UC Berkeley. The project will also enable postdocs and graduate students on the project to gain experience in training students from diverse backgrounds.Recent progress in genome editing technology is poised to accelerate plant breeding programs by allowing for the precise introduction of specific changes to important plant genes. Despite this advance, a primary bottleneck remains: fast and effective delivery of the gene editing reagents into crop plants. The most common methods of delivery are to encode RNA-guided genome editors (e.g. CRISPR-Cas enzymes) within transgenes and use tissue culture and transformation approaches or to introduce CRISPR protein and guide RNAs directly into plant cells followed by tissue culture to regenerate plants. However, tissue culture methods require considerable time, resources, and technical expertise, and can cause unintended changes to the genome and epigenome. Furthermore, regenerating plants from tissue culture only works in a limited number of plant species and genotypes. Plant viruses are ideal vectors for the delivery of CRISPR systems to whole plants without the use of plant transformation or tissue culture. However, most viruses have a very small cargo capacity, which is insufficient to accommodate currently used CRISPR systems. We propose to discover and characterize hypercompact CRISPR systems that are small enough to be encoded in plant viruses for easy and fast editing of whole plants, which could be used in a wide range of important crop species. We will screen a large number of candidate compact gene editing systems from metagenomics data and test these systems in bacterial cells and plant cells. The goal will be to find hypercompact editing systems that match or exceed the efficiency of current large systems.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.