Designing sgRNA Sequences for Therapeutic CRISPR Applications Targeting T790M and L858R Mutations in Lung Adenocarcinoma

Abstract

The formation of non-small lung cancer, particularly adenocarcinoma, can be attributed to several key mutations within the Tyrosine Kinase domain of the Epidermal Growth Factor Receptor (EGFR) gene. Said mutations result in a receptor that remains activated despite the absence of its ligand, continuously activating downstream oncogenic pathways, such as MAPK and PI3K/AKT, which in turn lead to uncontrolled division and autophagy. Two mutations in particular–exon 19 deletion (ΔE746-A750) (44%), and L858R (41%)–account for nearly all cases of EGFR-caused Adenocarcinoma. At the same time, secondary mutations such as T790M confer resistance to first-generation tyrosine kinase inhibitors (TKIs).  The use of the CRISPR process, along with the Cas9 enzyme, shows significant promise in cancer therapy. By employing bioinformatic platforms such as Benchling and CHOPCHOP, our team hypothesized that it is possible to design a single-guided RNA (sgRNA) that guides the Cas9 endonuclease towards specific oncogenes, allowing it to cleave these genes, thereby enabling targeted gene ‘knock-outs’. However, we found that this approach faces numerous limitations, including low on-target specificity, which compromises safety and precision.