Valencia_UPV

Description: The aim of this project is to develop a new and more efficient strategy for genome editing in plants using the CRISPR/Cas9 editing system. To reach this goal, we have made a Tool&Kit in order to decrease current technological barriers for plant breeders. It combines the split Cas9/CPF1 technology, viral systems, a CRISPR target database and laboratory equipment. Plant improvement with Agrobacterium - the most common method - is inefficient, long and difficult. The alternative chosen in our project are viral vectors, due to their higher rate of plant transformation. This advantage of virus is used to improve plants using CRISPR/Cas9 system. Nevertheless, viral vectors are small, so Cas9 and CPF1 endonucleases - necessary for editing - need to be splitted in two parts to fit into the viral vector. The reassembly of these parts is accomplished by inteins. In order to provide the necessary information to use the editing system, it has been created an optimized database that provides target genes and optimal gRNAs to knock-out them. This gene modification leads to a phenotypic improvement. A Testing System using Agrobacterium allows us to know how efficient is the gRNA selected in the database for any particular variety. To fulfill this process, we have made a Tool&Kit which includes all the necessary laboratory equipment for genome editing in a compact and affordable way, in addition to the technologies and techniques mentioned above. Together, they make an innovative and complete set of tools for accessible and more efficient genome editing in plants. Imagine a farmer who wants to have an orange tree that produces more oranges. But he knows that his variety tastes better than the ones offered by seedbeds. To get this goal he could cross varieties, but it takes a long time and it is expensive. The better way would be to genetically modify the plant. However, he doesn’t have the necessary knowledge nor the technology to make it possible. Plant breeders are the ones with the required knowledge to deal with his problem. They do have some of the necessary tools to make genetic edition, but they will need to invest a substantial amount of time and money to do it with them. With the current technologies the whole process will need between 2 and 12 years and around XX $ to develop an improved variety. Problem Currently, the most serious problems for plant breeders are time, money, and legal aspects regarding transgenic crops. That reduces the accessibility for those who want to modify the plant varieties. Using our new and revolutionary strategy based on CRISPR/Cas9 or CPF1 and viral systems, plant breeders will be able to modify any variety in order to get the desired features. Thus, those users could get their new crops spending less time and therefore less money. How will we do this? With HYPE-IT, a technology that will allow to Hack Your Plants Editing with the tools created by our team. We pretend to bring this new technology from laboratories closer to land. We will create an optimized database containing target genes whose silencing with gRNA lead to a phenotypic improvement. The plant breeder will select in our database the desired modification or gene to be edited for his variety, and it will return the optimal guide RNA to target and knock-out the selected gene. Open source will be the main philosophy of this database, allowing breeder to update new known modifications. The plant breeder could test the guide RNA directly in his plant, but it would take a long time just to check if it works or not. To avoid this, we have designed a gRNA Testing System which does this in a fast and simple way. Using Nicotiana benthamiana, Agrobacterium infection system, a short sequence of the gene of his plant and a luciferase assay, the users will surely know if the guide RNA works on his own variety. To genetically modify the plant, the Agrobacterium infection system is inefficient , long and difficult. Thus, we decide to use a viral system to enhance plant infection. Since the viral vectors are small, the Cas9 and CPF1 endonuclease - necessary for editing - need to be splitted in two parts. To reassemble them inside the plant cells, we will use intein proteins. All these innovative tools can overcome the problems that plant breeders have nowadays. Bearing this in mind, we want now to make genetic plant edition more accessible for all potential users. We have made a Tool&Kit that contains all these technological tools. For users who have few resources we have included all the laboratory equipment required to make the genetic edition, with simple protocols.
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Year: 2016Visit Wiki
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Updated at: 8/9/16