Utilization Of CRISPR/Cas9 For The Protection Of Crops

The protection of crops has been an ongoing battle. Genetic engineering introduces new strategies to control pests. These new strategies produce transgenic plants that contain insecticidal traits. One of the best defences against plant eating insects is Bacillus thuringiensis (Bt). Bt is naturally found in soil. Bt can be genetically engineered into a plant which will then enable to plant to produce its’ own Bt crystal protein (CP). The CP is toxic to insects of concern. The insects will have to feed off the plant and ingest the CP, once this happens the CP is activated inside the gut of the insect and the insect will die of starvation or infection. Bt is registered to us by the US Environmental Protection Agency (EPA) since 1961. Insects that are known to feed on spinach are: caterpillars, wireworms, crown mites, aphids and leafminers.

The main insect that will be targeted is caterpillars, to target caterpillars the Bt subspecie kurstaki will be used. The gene in Bacillus thuringiensis that produces that CP gene will be used and inserted into spinach plants so that the plants will be resistant to caterpillars. The CP gene will be isolated from Bacillus thuringiensis. This gene will then be inserted into an empty gRNA Expression Vector. These plasmids contain both the gRNA and Cas protein. In this case the gRNA will contain the CP gene. I found a plasmid that can work for both monocot and dicot pants, this plasmid contains a PCR-based procedure which can rapidly generate multiple gRNA expression cassettes. These cassettes can then be assembled into binary CRISPR/Cas9 vectors through cloning by Golden Gate ligation of Gibson Assembly. The plasmid I found is called the pYLsgRna-ATU6-29 (a figure of the plasmid is provided below).

The CP gene will thus be amplified using PCR-based methods, this gene will then be cloned into a binary CRISPR/Cas9 vector using Gibson Assembly. Once the plasmid is constructed it should contain the gRNA which contains the CP gene as well as a Cas9 enzyme. The plasmid can ten be inserted into a phage. The phage will then infect the host plant (spinach) and insert the plasmid into the spinach cells. The cells will then respond using their natural CRISPR technology and the CP gene will be inserted into the host (spinach) genome as a spacer. Homology-directed repair (HDR) will be used to knock-in the CP gene. This repair system can insert sequences by using recombination of the target locus with the supplied DNA as a donor template.

The CP gene will thus be incorporated into the host genome and the host will produce the CP toxin which will cause the death of caterpillars once ingested. The selection for the edited genomes will occur since an ampicillin resistance gene is also transformed. In The initial steps of this process all the untransformed phages will die as they will not be able to survive in the presence of ampicillin, thus all the phage’s that will be used will contain the CP gene.