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HYPE-IT by Valencia UPV iGEM2016

Nowadays, the most important challenges to society are providing food, energy and other materials from limited natural resources. Plant Synthetic Biology has sprouted as the revolutionary method to solve these problems by designing and developing new plant devices. In the upcoming years this new scientific field, that gathers engineering principles along with plant biology, is going to emerge as one of the mainstays of agriculture through improvement of traditional crops.

Working with plants is a challenge. Public perception of synthetic biology and genetic modification is one of the biggest difficulties to be faced. Furthermore, technical obstacles such as the lack of resources and funding in basic plant research limits accessibility to the benefits of these technologies.

HYPE-IT

hypeitworkflow
HYPE-IT workflow

Bearing this in mind, our goal is to improve the accessibility of genome editing in plants with CRISPR/Cas9. This strategy combines a split Cas9 variant with viral vectors to make gene knock-outs. An optimized CRISPR/Cas9 target database has been created to search modifications of interest. To know the efficiency of the gRNA obtained from the database, it will have been designed in a Testing System. These tools are gathered together with the laboratory equipment (Lab-case) necessary to make genome editing. As an affordable and simple product, it will help to decrease current technological barriers for plant breeders.

Over the next years, the exponential increase of population will make necessary to increase and enhance agricultural production all over the world. Nowadays, obtaining enhanced plants takes a long time and it is expensive.

The best way would be to genetically modify plants, since other methods are inefficient or dangerous, such as irradiation with cobalt isotopes. However, farmers don’t have the necessary knowledge or the technology to do this. Plant breeders are the ones with the required knowledge to deal with this problem, but they need to invest a substantial amount of time and consequently money to do it with the current techniques. Given that, we strongly believe that the solution is to make genetically improved crops accessible to everyone.

Therefore, we propose using genome editing tools based on the revolutionary system CRISPR/Cas9-CPF1. Plant breeders could improve any variety with the features that farmers wished. How will they do this? With HYPE-IT, an innovative technology that allows to ‘Hack’ Your Plants Editing with the tools created by our team. We aim to bring this new technology from laboratories closer to land.

Split cas9

splitcas
Split Cas 9. Source: Nucleic Acids Res, 43(13), pp.6450-6458.

 

To improve a desired plant variety, we have decided to use a non-pathogenic viral system, which enhances plant infection compared to the conventional Agrobacterium infiltration system. Agrobacterium-mediated plant transformation is a highly complex process that relies on genetic determinants of bacterium and the host plant cell. It has the disadvantage of being inefficient, not all plants are susceptible to common Agrobacterium strains and it is slow, requiring at least two years depending on the species to obtain the modified plant without transgenes. Additionally, the level of transgene expression in plants is highly variable. Often, lines of transgenic plants obtained with Agrobacterium-mediated modification lose the modifications after several generations of growth under field conditions. We need to develop strategies to enhance the extent and stability of transgene expression. For that reasons, the viral system has been chosen as the best alternative to Agrobacterium. However, viral vectors are small and don’t accept big insert sizes. For that reason, the Cas9 and CPF1 endonucleases – necessary for editing – will be split in two parts. They will be joined inside the cell using inteins. CPF1 is used as an alternative for Cas9, as it recognizes different regions in the DNA.

 

Target database

We will create a database containing target genes whose silencing with gRNA leads to a phenotypic improvement. Open source will be the main philosophy of this database, so anyone could upload new genes, modifications and possible variations. Furthermore, it will be connected with other public databases and references such as NCBI or Solgenomics, to make a better searching tool. The database will also include an internal software which provides an optimal guide RNA to target and knock-out the selected gene, so the user does not need to design it.

This database with an integrated processing software is designed specifically for plant breeders. They may or not know the gene that they need to knock-out in order to obtain the desired phenotypic modification.

 

gRNA Testing System

Testing the predicted guide RNA directly in the desired variety takes a long time. To avoid this, we have created a gRNA Testing System, so the users will know in a few days if the guide RNA will work in their variety. We will do this using Nicotiana benthamiana, because of its fast growth rate. Also, we use Agrobacterium infection system, to obtain accurate results using a simple method, a short sequence of the gene of his plant, and a luciferase assay. The infection system with Agrobacterium, although being inefficient for genome editing, is effective for transient expression, which is enough to test the gRNA.

At first, the luciferase reporter gene will be out of frame. When CRISPR-Cas9 system acts, it produces indels. These additions and removals of bases will change the reading frame. Our system is designed to allow the transcription of the luciferase when the modification occurs, because the gene will be in the correct reading frame and it will be translated correctly. In that case, plant breeders could see in a simple way if the genome editing has worked.

luciferasegrnatestingsystem
luciferase grna testing system

Hardware and reagents

The plant breeder has a database to choose the genes to knock-out, a predicted gRNA for that genes, a testing system to be sure this gRNA will work. Also, they have the optimal vehicle -viral vectors- to infect the plant with split Cas9 and the gRNA. This would be enough for a plant breeder who has access to a fully equipped laboratory. Nevertheless, we want now to make plant gene editing accessible for all potential users, even if they do not have an advanced laboratory. To achieve this goal, we have made a hardware set up which contains all the tools with the necessary reagents required to perform genome editing, with simple protocols. This guarantees availability for agricultural cooperatives, start-ups, garage laboratories…

This will be the future of agriculture: making genome editing of plants accessible to everyone.

hardware
HYPE-IT Hardware design

Our team
Valencia UPV team has been participating in the iGEM competition for 10 years. The last three projects have been based on plants, in collaboration with the Plant Molecular en Cellular Biology Institute (IBMCP) and the Superior Council of Scientific Research (CSIC).
This year the team is composed by 9 students of the Polytechnic University of Valencia (UPV): biotechnologists, computer scientist and biomedical, electronic and industrial engineers. Our project is motivated by both the exciting idea of using the innovative CRISPR/Cas9 system and the need to improve genome editing. The latest is a technology which will be a must in our future, and we thought it was necessary to improve it in a way that allowed new varieties to reach the market sooner.

Our team is really engaged in this fascinating project. If you’d like to know more about it or you are interested in supporting us, email us at valencia.upv.igem@gmail.com. You can also follow us on Twitter (@UPVigem) and Facebook (Valencia UPV iGEM).

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