Document Type : Original Article

Authors

• Faezeh Sabzehei ¹
• Shirin Kouhpayeh ²
• Mansoureh Shahbazi Dastjerdeh ¹
• Hossein Khanahmad ³
• Rasoul Salehi ¹
• Shamsi Naderi ¹
• Razieh Taghizadeh ¹
• Parisa Rabiei ¹
• Zahra Hejazi ¹
• Laleh Shariati ¹

¹ Department of Genetics and Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran

² Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

³ Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan; Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Noncommunicable Disease, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Background: Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods. Materials and Methods: At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F' that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry. Results: The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) (P < 0.0001). Conclusion: According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample.

Keywords

• Gene editing tools
• green fluorescent protein expression system
• zinc finger nuclease