Bernardo-Ferry Abner
Research Mentor: Xin Tong
Mentor Department: Molecular & Integrative Physiology, Medicine
Author(s): Bernardo-Ferry Abner, Eric Jaffee, Xin Tong
Session: Session 1 (9:00 AM – 9:50 AM)
Presentation Type: Poster 6
Abstract
Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is a prevalent chronic liver disease with severe outcomes, including liver failure and liver cancer, and is projected to increase significantly in the coming decades, with prevalence in the United States expected to rise from 33.7% of adults in 2020 to 41.4% by 2050 (Le). Given this growing clinical burden, our lab is investigating the multifaceted function and regulation of Carbohydrate Response Element-Binding Protein (ChREBP), a lipogenic transcription factor that senses sugar intake and activates genes involved in carbohydrate and fat metabolism, particularly in the liver during diet-induced metabolic dysfunction-associated steatohepatitis (MASH). To better understand how ChREBP contributes to disease progression, this study aims to develop a hepatocyte-specific gene knockout model using CRISPR-Cas9 gene editing. Two guide RNA (gRNA) sequences targeting the 5’ region of the mouse ChREBP gene were designed and cloned into the pX602 AAV plasmid, which expresses the Staphylococcus aureus Cas9 enzyme under a hepatocyte-specific TBG promoter and drives gRNA expression via the U6 promoter. Following cloning, mini culture clones will be screened using restriction enzyme digestion and verified through Sanger sequencing to confirm successful insertion of the gRNA sequences. Verified plasmids will then be used to generate AAV viral particles through transfection of HEK293 packaging cells alongside helper plasmids, after which viral stocks will be purified, concentrated, and quantified using Coomassie staining and qPCR. Finally, the AAV virus will be delivered to wild-type mice via tail vein injection, and the efficiency of ChREBP deletion will be assessed through DNA, RNA, and protein analyses. This approach will establish a precise in vivo gene-editing model and provide insight into the molecular mechanisms by which hepatic ChREBP contributes to the development and progression of MASH.
