Targeted Knockout of F9 by CRISPR-Cas9 and Establishment of an In Vitro Hemophilia B Model in HepG2 Cells
Project no. 23766 · Status: Kapanmış (Yayın bekleniyor) · 2022
Summary
<p>Hemophilia B is an X-linked inherited bleeding disorder caused by pathogenic F9 variants that reduce the level or activity of coagulation factor IX (FIX). This project aimed to disrupt F9 in a targeted manner using CRISPR-Cas9 and to establish a HepG2-based in vitro cellular system for investigating FIX loss of function. Three sgRNAs targeting Exon 2 and Exon 5 were designed using the F9 reference transcript NM_000133.4, generated by in vitro transcription, and screened for cleavage with recombinant SpCas9. The corresponding sequences were cloned into pGuide-it-sgRNA1 and verified by Sanger sequencing. A Gesicle Producer 293T-based nanovesicle system was used for transient delivery of Cas9-sgRNA ribonucleoprotein complexes to HepG2 cells. Target-locus Sanger analysis supported editing-associated sequence changes. FIX activity measured 1.0% in HepG2 conditioned medium before editing and 0.2% after Exon 5-targeted RNP treatment; because serum-free DMEM also yielded a 0.2% background value, the functional data support a marked reduction in FIX activity but do not by themselves establish a complete null phenotype. The findings support an F9-loss-of-function, polyclonal HepG2 model for Hemophilia B research. Confirmation of a fully null genotype will require single-cell cloning and quantitative, allele-resolved genotyping.</p>