Use of CRISPR-Cas9 Gene Editing for Generation of Mutant Yeast for PTC Readthrough

Emily Gaines, Dr. John Hartman IV, Department of Genetics, University of Alabama at Birmingham, 1720 University Blvd Birmingham AL 35294

The CFTR (cystic fibrosis transmembrane conductance regulator) gene encodes a polytopic ABC transporter that is trafficked to the plasma membrane where it functions to help maintain the balance of salt and water in many organ surfaces of the body. Mutations in the CFTR gene can cause reduced or complete loss of function which leads to decreased airway surface liquid and thickening of epithelial mucus in organs, particularly in the lungs and pancreas, resulting in cystic fibrosis pathology.

The purpose of my research in the Hartman laboratory is to explore CRISPR-Cas9 gene editing techniques in yeast to model mutations found in CFTR using the gene most homologous to CFTR in yeast: YOR1. Current research is to adopt a technique developed in the Marcotte laboratory, using yeast plasmids to express Cas9 and CRISPR guide RNA along with co-transfected homology dependent repair oligos to introduce targeted mutations. Using this technique to generate mutations that result in premature translational termination (a.k.a., ‘stop’, ‘PTC’, or ‘X’ mutations) of Yor1, analogous to disease mutations in CF patients, will allow us to characterize genetic mechanisms of PTC readthrough, and their therapeutic potential. 

tRNA readthrough would allow the ribosome to continue reading past the stop codon on the mRNA strand to the next stop sequence. The lab hypothesizes that via the wobble effect, where the third base pair can vary without affecting the production of a protein, a tRNA can also be mutated to read through the various stop codons that cause no production of vital proteins and cause cystic fibrosis.

Additional Abstract Information

Presenters: Emily Gaines, Ryan Mancinone

Institution: University of Alabama at Birmingham

Type: Poster

Subject: Biology

Status: Approved

Time and Location

Session: Poster 2
Date/Time: Mon 3:00pm-4:00pm
Session Number: 2582