Cas9 in Plants Project

A CRISPR/cas9 system is a large protein guided by a self-guiding RNA, which is capable of targeting specific DNA sequences. Cas9 has been characterized previously in bacteria and mammalian cells. Often targeted to a promoter region, Cas9 acts as highly effective gene repressing tool. The goal of the cas9 project is to make this regulatory tool available to plant genetic engineering.

Introduction

A CRISPR/Cas9 system, which is found in a few prokaryotic organisms allows for very specific DNA targeting. The system as found is used to cleave DNA or completely cut out certain sections, perhaps as a defense mechanism. However, scientists in recent years have turned their attention to CRISPR/cas9 systems due to the highly specific targeting abilities. Mutation of a few key areas of the protein caused the creation of dcas9. dcas9 is catalytically inactive and can be expressed in controlled quantities and will not cleave DNA. When coupled with a 100bp engineered self-guided RNA sequence the dcas9 protein can be bound to any 20bp DNA sequence. This has many implications including gene-specific down regulation, localization abilities via attachment to the cas9 protein complex, and gene-sepcific up regulation via the attachment of an enhancer targeted to a promoter region. There are many possible uses of dcas9, and would serve as an excellent new tool in the plant synthetic biology toolkit.

Background

The below diagram describes how the dcas9-guided RNA targeting system works.

Reference: http://www.sciencedirect.com/science/article/pii/S0092867413002110

Method

To test dcas9 we designed a vector that expressed both GFP and RFP under two different CMV35s promoters. Transforming this plasmid into plants should show a constant GFP to RFP ratio under florescence spectroscopy.(this vector is identical to the one designed for the promoter project) Once this generic ratio is established we can modify this plasmid to include the dcas9 protein and guiding RNA sequence. If we target the guiding RNA to the GFP/promoter polymerase binding region we should expect a ratio indicating a decrease in GFP expression. It should be noted that dcas9 and the RNA guiding sequence will both need their own unique promoters since plants have a tendency to destroy multiple promoter regions.

Further Study

Due to limitations resulting from the lack of previous synthetic biology application within plants, we were unable to obtain any results. We have successfully isolated the desired parts needed for assembly of the plasmid and have the backbone with the 35s promoter left border and Nos terminator right border. Successful construct assembly of these parts is still needed for bacterial and plant transformation. Following promising transformation, fluorescence testing and characterization must be done.

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