In Silico Design and Evaluation of a CRISPR dCas9 DNMT3A DNMT3L Epigenetic Editing System Targeting the CDKN2A Promoter
CDKN2A (p16) is a tumor suppressor gene whose normal function is to stop the cell cycle from over dividing, allowing cells to enter senescence after completing their function. When the CDKN2A promoter is methylated, the gene is silenced, and cells continue dividing rather than stopping, a mechanism shown in published research to delay cellular senescence and support extended cell culture, with relevance to stem cell research. This project used a CRISPR based dCas9 DNMT3A DNMT3L epigenetic editing system to computationally design, filter, and evaluate candidate single guide RNAs targeting the CDKN2A promoter. Using CHOPCHOP, 284 candidate guides were generated and narrowed to six based on GC content, self-complementarity, and zero perfect match off targets, then independently verified using Cas-OFFinder, resulting in three final candidates with zero off target hits across all tested mismatch levels. The UCSC Genome Browser confirmed that all three final guides fall within a continuous CpG island overlapping active regulatory elements. Structural visualization of published crystal structures (PDB: 4UN3 and 5YX2) illustrated the roles of the targeting and catalytic components of the fusion protein. Methylation efficiency for each guide was estimated by comparison to published experimental results, yielding estimated ranges of 25 to 40 percent, 20 to 35 percent, and 15 to 30 percent for the three respective guides. These estimates require experimental validation through bisulfite sequencing before any conclusions can be drawn about actual silencing capacity. This project demonstrates a complete computational workflow for CRISPR based epigenetic editing design, grounded in published experimental precedent.