Andrei Vava
Research Mentor(s): Sebastian Vishnopolska
Mentor Department: Department of Human Genetics
Authors: Andrei Vava, Sebastian Vishnopolska
Session: Session 6 (3:00pm – 3:50pm)
Presentation Type: Poster 25
Abstract
The gene ARID1A (AT-rich interaction domain 1A) encodes an essential subunit of the SWI/SNF chromatin remodeling complex. Mutations in ARID1A are highly prevalent in individuals with endometrial cancer and cancers of the digestive and urinary tracts. In the COSMIC database, single nucleotide variants (SNVs) are the most frequent ARID1A mutations identified in cancers, particularly nonsense and missense variants. However, most of these variants have unknown significance, meaning that their particular impact on protein function, and therefore their relationship to cancer development, is unknown. Prime editing is a gene-editing tool designed to introduce specific changes into the endogenous DNA sequence of a cell. The editors consist of a fusion of a Cas9 nickase and a reverse transcriptase, paired along with a prime editing guide RNA (pegRNA) that specifies both the target site and the desired change. We tested three different prime editor protein complexes, PE2, PE4, and PE7 to determine their efficiency in creating desired three-base insertions in HAP1 cells and found that PE7 shows a tenfold increase in efficiency compared to PE2 and PE4, and therefore we used this editor in subsequent experiments. We selected a handful of ARID1A SNVs reported in the ClinVar database with different pathogenicity classifications (benign, pathogenic, and uncertain significance), and designed pegRNAs to model each in HAP1 cells. Next, we seek to test the functionality of each variant by its resistance to 6-thioguanine (6-TG), which our lab has previously shown to be a predictor of loss of ARID1A function. We hypothesize that pathogenic missense variants will act in a similar manner to truncating mutations while benign variants remain susceptible to 6-TG treatment, enabling us to assess the pathogenicity of reported and future variants of uncertain significance in this gene.
