The CRISPR/Cas9 system can be used for gene editing purposed including inserting genes, deleting genes, and creating breaks in the DNA.
Predicted CRISPR/Cas9 System
In our first three test groups, we will only be using one guide RNA for each cell culture. The CRISPR/Cas9 will create a double strand break at the target sequence, leaving the ecDNA in two pieces. breaks in the DNA.
In our 4th test group, we will be using 3 guide RNAs for each cell culture. This will create double strand breaks at 3 target sequences, leaving the ecDNA in 6 pieces. We would like to test this because the more breaks we create in the ecDNA, the harder it is for the ecDNA to ligate back together.
Predicted & Modeled Transfection Results
Based on previous CRISPR/Cas9 knock out procedures, ecDNA cell count is predicted to considerably reduce(10-20%) after transfection procedures. The broad estimations of the reductions of cell growths and cell growth rate lead to the hypothesis that the modification of ecDNA could result in exponential reductions of rates compared to control along a short time frame. After the transfection procedure on ecDNA cells, experimental groups are expected to react to a different degree based on the individual guide RNA. This should result in distinctive, yet statistically significant experimental group cell count reduction over time.