Farzad Soheilipour - Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Department of Biology, Faculty of Basic Sciences, Gonbad Kavous University, Gonbad Kavous, Iran.
Sohrab Boozarpour - Department of Biology, Faculty of Basic Sciences, Gonbad Kavous University, Gonbad Kavous, Iran.
Shiva Aghaei - Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Ehsan Farashahi Yazd - Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Background: Stable Cas۹ (CRISPR-associated protein ۹)-expressing cell lines have emerged as valuable tools in genetic research, enhancing the efficiency of the CRISPR/Cas۹ system and streamlining gene editing procedures. These cell lines enable simultaneous editing of multiple genes and reduce the overall editing time. Objective: This study aimed to develop a stable human fibroblast cell line capable of genetic conversion into a mutant form, serving as a cellular model for a specific genetic disease. The established cell line facilitates investigation of disease mechanisms, testing of potential treatments, and gaining insights into underlying molecular processes. Materials and Methods: Human embryonic kidney ۲۹۳LTV cells were used to produce pseudo-virus particles, while Yazd human foreskin fibroblasts batch ۸ (YhFF#۸) cells were targeted for genetic modification. Transfection of human embryonic kidney ۲۹۳LTV cells with pCDH-Cas۹ plasmid DNA generated pseudo-viral particles. YhFF#۸ cells were transduced and selected using antibiotics. Green fluorescent protein (GFP) detection confirmed successful transduction and selection. Relative expression levels of the Cas۹ gene were determined by quantitative polymerase chain reaction. Results: The study validated the fidelity of the Cas۹ gene cassette sequence and its transcriptional activity. Transduced YhFF#۸ cells exhibited green fluorescence, with antibiotic selection resulting in nearly ۱۰۰% transduced cells. A reporter GFP gene enabled real-time monitoring of YhFF#۸-Cas۹-GFP-PuroR cells using fluorescence microscopy. Conclusion: YhFF#۸-Cas۹-GFP-PuroR cells, labeled and susceptible to genomic editing, provide an optimal source for generating induced pluripotent stem cell lines for future biomedical research.

Fibroblasts, Cell line, Genetic transduction, CRISPR-Cas۹., فیبروبلاست ها, دودمانه سلولی, ترانزداکشن ژنتیکی, کریسپر-کاس ۹.