Non-Viral Delivery of CRISPR-Cas 9 in Neuro-2a Cells for the Stable Production of Natural Products

Neurological cells tend to be more resistant to gene editing techniques, but if gene editing is possible in neurological cells it could increase therapeutic solutions for neurological diseases such as Alzheimer’s disease, Parkinson’s disease or brain tumors. Specifically for Alzheimer’s disease, the introduction of curcumin to the cell decreases the effects of the disease. Non-viral delivery of a plasmid encoding for the enzymes tyrosine ammonia lyase (TAL), 4-coumarate-CoA-ligase (4CL1) and curcuminoid synthase (CUS) would facilitate transformation of tyrosine into bisdemethoxycurcumin (ddCCM). The stable expression of this process using this plasmid and a Cas 9 plasmid in Neuro-2a cells can be used to study the effects of curcumin against β-amyloids associated with Alzheimer’s disease. Succinylated polyethylenimine has been shown to increase transfection efficiency in serum and therefore can be used to insert these two plasmids into the Neuro-2a cells for analysis. The transfection efficiency of succinylated polyethylenimine in Neuro-2a cells was studied using pGL3 firefly luciferase to determine the optimal polyplex. From this analysis, the 2%, 3% and 6.5% succinylated polyethylenimine demonstrated the best transfection in serum. These polyplexes were then used to transfect the Neuro-2a cells with the TAL, 4CL1 and CUS plasmid. Stable expression was analyzed using gas chromatography. Cell viability was tested using cell titer blue and an ROS assay. The addition of the TAL, 4CL1 and CUS enzymes facilitated production of ddCCM which increased cell viability when exposed to β-amyloids.