Generation of CRISPR knockout models of heterozygous hepatocyte nuclear factor-1β (HNF1B) towards understanding kidney diseases

Abstract

Hepatocyte Nuclear Factor 1 Beta (HNF1B) is a DNA-binding transcription factor that is essential for normal kidney development and is expressed in all tubular epithelial cells composing the nephrons and collecting ducts where it controls the expression of genes involved in membrane transport, cell differentiation, and metabolism. In this study, CRISPR/Cas9 technology was utilised to generate HNF1B gene knockout in HEK 293T cells, to investigate the effects of HNF1B gene loss. To achieve this, HEK 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin (10,000 IU). Two guide RNAs (sgRNAs) targeting HNF1B gene were designed using the CRISPick web tool, and DNA breaks were repaired through non-homologous end-joining (NHEJ) pathway. The sgHNF1B was cloned into the pKLV-U6gRNA(BbsI)-PGKhygro2ABFP vector, transformed into E. coli and successful cloning was then validated using Sanger sequencing. Post-transduction of Cas9 lentiviral construct, cells were selected with 30 μg/mL Blasticidin antibiotic, and the edited cells were isolated. Subsequently, these cells (containing Cas9) were transduced with sgHNF1B lentivirus and further selected using 700 μg/mL Hygromycin selection for 6 days and continue with western blot to validate the protein expression. The western blot band intensity analysis indicates that HNF1B protein expression is reduced in both knockout cell lines, KO1 (60%) and KO2 (34%), compared to untransduced control (100%). The observed reduction in protein expression levels suggests that the knockout is heterozygous and not full knock out. This is likely due to a mixed cell population, where some cells are fully edited (bi-allelic KO), others are partially edited (monoallelic) and some may remain unedited resulting in intermediate levels of protein expression when analysed collectively. The HNF1B gene knockout cells were successfully compared with untransduced control cells and further validated using western blot analysis confirming the HNF1B gene knockout at the protein level. In conclusion, this study had successfully generated heterozygous HNF1B CRISPR-knockout models of kidney dysfunction as a proof-of-concept that gene function can be altered to treat kidney diseases. In addition, this study also highlights the utility of genome editing as a powerful tool for elucidating gene function and effectively modelling human pathophysiological conditions. Through this CRISPR/Cas9 technology, valuable insights are offered to drive advancements in personalized medicine and sustainable healthcare solution. Such innovations address key biomedical challenges and advance scientific progress to improve the well-being of both individuals and the planetary health. Keywords: CRISPR/Cas9, Gene editing, HEK 293T, HNF1B, sgRNA cloning, kidney