Engineering viral vectors for high-throughput CRISPR genetic screens in plants

Tools based on CRISPR-Cas systems hold great potential both for fundamental plant genetics and precision crop breeding. Unlike traditional mutagenic approaches, CRISPR-Cas systems allow for the simultaneous delivery of thousands of unique guide RNAs, enabling the generation of collections of higher-order mutants for rapid functional screens and the discovery of genetic interactions. However, CRISPR technology suffers from a ‘transformation bottleneck’; relatively few crop genotypes can be transformed and often only a handful of mutant lines can be produced. In virus-induced genome editing (VIGE), plant viruses are engineered to deliver gRNAs into cells. Such a capability is highly attractive for CRISPR knock-out (KO) screens as it could simplify the delivery and generate large populations of mutant lines in a broader range of crop genotypes. However, the application of VIGE for CRISPR KO screens is hampered by the lack of a proper viral-based multiplex gRNA delivery vector. My host lab has pioneered the development of CRISPR KO screens in plants, and in VISCREEN I will combine this knowledge with my expertise on VIGE to scale up CRISPR-Cas-mediated manipulation of plant genomes. The main objectives of my project are: (i) to develop Barley stripe mosaic virus (BSMV) for high-throughput CRISPR screens in wheat and maize; and (ii) to engineer and validate a set of viral vectors for VIGE in soybean. The viral vector-based, high-throughput editing systems developed in VISCREEN will expand CRISPR screens in a wider variety of crops and allow to test fundamental biological questions that are limited by functional redundancy. Overall, this innovative approach will make me a well-trained academic researcher with the capabilities to develop a professional career within the plant genome editing field.