Poster X12 - Formulation of CRISPR Cas-9 loaded polymer mesoscale nanoparticle

Introduction: CRISPR associated protein Cas-9 system, a unique gene editing system can be used for various applications. It has shown potential application in different types of cancer including kidney cancer. They can be used as a therapeutic to inhibit growth of cancer cells and tumors because previous studies have shown knockouts and knockdowns in tumor suppressing gene associated to kidney cancer. Since it is a system prone to degradation different vectors are used for its delivery to the target mutated gene. Mesoscale nanoparticles (MNPs), highly biocompatible, are known for targeting the renal tubules and their controlled release. They also can overcome multiple barriers and limitations of viral delivery systems. In this study we aim to knockout the green fluorescent protein (EGFP) from renal cancer cells using CRISPR Cas 9 loaded MNPs and hopefully correct mutations with high precision and thus reduce the progression of the kidney disease which involve gene alteration and chromosomal aberrations.

Materials and

Methods: In this work, we have synthesized polymer MNPs with EGFP single guide RNA (sgRNA) and Cas9 protein or a ribonucleoprotein (gRNA and Cas9 together) using nanoprecipitation. The loading efficiency of the gRNA and the protein was studied using Quant iT Ribogreen assay and Quant iT protein assay respectively. In vitro studies were done on renal carcinoma cell lines transfected with EGFP and were used to test the nanoparticles capability of knockout or knockdown. Imaging was done using fluorescence microscopy and quantitative analysis was done using Image J software. Flow cytometry was also performed for the analysis of fluorescence levels in the cells after treatment. Furthermore, we are performing polymerase chain reaction (PCR) on the extracted genomic DNA or plasmid DNA and running gel electrophoresis to determine the plasmid DNA in the sample and then proceed towards sequencing.

Results, Conclusions, and Discussions: The fluorescent microscope images showed a decrease in fluorescence for the cells treated with the CRISPR Cas9 particles. Image J analysis of these cells also showed a difference between the treated cells and the non-treated ones. The flow cytometry experiments showed a shift in the fluorescence peak for the CRISPR Cas9 treated cells, but it also showed a shift in the non-target gRNA and Cas9 MNPs treated cells. We are anticipating that this might be due to off-site targeting. However, when we tested a similar experiment with empty MNPs there was no shift in the peak. This can be further confirmed after sequencing experiments are completed. After the successful completion of the proof-of-concept study we are aiming towards a disease model particularly targeting the kidneys and hope to use CRISPR Cas 9 specific MNPs in the treatment of the same.

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