HYPE-IT (Hack Your Plants Editing with Innovative Technologies) main goal is to develop an affordable technology for improving the accessibility of genome editing in plants with CRISPR/Cas9. Our strategy is based on a split Cas9 variant with viral vectors with the purpose to make gene knock-outs, leading in new enhanced plant varieties. A gene Database has been created, gathering identified referenced genes, in addition to more necessary information. There, we have related gene sequences with an specific phenotypic trait. Affordable laboratory equipment, such as thermocycler or centrifuge, as well as all the necessary biological parts, has been designed. All these tools and reagents have been gathered under a Lab-case. It has all the necessary things to make plant genome editing as an affordable, simple and accesible product, decreasing current technological barriers for plant breeders.
One of the key elements in our project is the split-Cas9 system, designed to allow the delivery of Cas9 in plants with efficient viral vectors. To allow the reconstitution of the divided Cas9, he have used the inteins of 2014 Heidelberg Team (BBa_K1362400 and BBa_K1362401. We have improved its characterization by demonstrating its functionality in plants (read more about Split-Cas9).
We have designed a Workflow, where we illustrate how a plant breeder could obtain his desired improve variety by HYPE-IT system:
- The plant breeder access to HYPE-IT Database to choose the genes to knock-out. The only thing he has to decide is what phenotypic trait he wants to obtain, as well as the species that he is going to work with.
- HYPE-IT Software give him predicted gRNAs for the targeted gene.
- These gRNAs are ordered by a scoring system, from the optimal one to the less accurate. Then, he order the gRNA synthesis.
- If he wants to be sure the gRNA is properly designed, a luciferase based gRNA testing system has been designed to check if this gRNA will work before its integration in the targeted plant.
- After that, split Cas9 infiltration is delivered by viral system.
- He waits until the plant grows enough.
- Then, he regenerates new plants from modified plants.
- These plants are regenerated from modified cells, so the whole plant is edited.
- The plant breeder harvest modified fruits.