Avery Hinojosa
Research Mentor(s): Mark Slayton
Mentor Department: Internal Medicine, Hematology & Oncology
Authors: Avery Hinojosa, Madison MacLachlan, Mark Slayton, Sultan Ahmed
Session: Session 3 (11:00am – 11:50am)
Presentation Type: Poster 12
Abstract
Non-small cell lung cancer (NSCLC) driven by the EML4-ALK fusion mutation has posed significant challenges in treatment due to its emergence of chemotherapy mutations. There is also a lack of available model cell lines that replicate these mutations in wet lab research, thus limiting the testing being done to overcome these mutations. Our approach utilizes a new technology, PASTE editing, which uses an engineered enzyme trio of CRISPR-Cas9, reverse transcriptase, and integrase for precise genome editing with insertions of 35 kilobases or more. PASTE editing requires multiple parts (plasmids) to function. Researchers who invented PASTE editing provided the plasmid encoding for the editor enzyme, but we need to tell it where to edit. To do this, we made plasmids containing guideRNA to target the normal EML4-ALK gene and a “cargo†plasmid that contains the modified EML4-ALK gene with the mutation. Using the PASTE enzymes, the genome is cut on the coding strand to allow integration of the cargo. To help ensure a higher success rate, we include a nicking guide that cuts open the opposing strand of DNA. This process tricks the cellular DNA repair system such that it doesn’t know which strand was edited. Therefore, it has a 50/50 chance of repairing the side that had our insertion, as opposed to a 10% success rate with one side only being cut. Our preliminary work has focused on assembling the necessary components, including the EML4-ALK cargo plasmids. Additionally, we have created nicking guides and guideRNAs that hold the correct genes for nicking/guiding where we want. The next steps involve PASTE editing the genomes of different cell lines and testing various chemotherapy drugs to assess their efficacy. This approach holds promise for creating more accurate models of NSCLC, while also advancing our understanding of chemotherapy resistance.
