Journal of Advanced Research in Applied Sciences and Engineering Technology 43, Issue 2 (2025) 178-188 178 Journal of Advanced Research in Applied Sciences and Engineering Technology Journal homepage: https://semarakilmu.com.my/journals/index.php/applied_sciences_eng_tech/index ISSN: 2462-1943 The Construction of CRISPR-Cas9 System Targeting Vector for CYP3A4 Gene in Hepatic Cell Lines Fazleen Haslinda Mohd Hatta 1 , Fazlia Natasha Abdul Aziz 1 , Shafiq-ur-Rehman 2 , Ahmad Azani Othman 1,* 1 2 Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), 42300 Bandar Puncak Alam, Selangor, Malaysia Institute of Microbiology and Molecular Genetics, University of The Punjab, New Campus Lahore, 54590, Pakistan ARTICLE INFO ABSTRACT Article history: Received 16 June 2023 Received in revised form 23 September 2023 Accepted 1 March 2024 Available online 17 April 2024 Drug metabolism in the human liver initiated by Cytochrome P450 (CYP450) has been widely acknowledged. They oxidise drugs, harmful compounds, and endogenous molecules like steroids. In our research study, we mainly focused on CYP3A4 of the CYP450 subfamily that is widely available in the liver around 15% - 20% of hepatic content and plays a significant function in metabolising up to 50% of drugs in the market today. However, the CYP3A4 gene encodes CYP3A4 enzymes that are highly polymorphic, which could affect enzymatic activity and cause variable responses in drug metabolism. Clustered Regularly Interspaced Short Palindromic Repeats Associated Protein 9 also known as the CRISPR-Cas9 system is a gene editing method that allows scientists to edit parts of the genome by insertions and deletions. Hence, this method would be useful for genetic variants such as the CYP3A4 gene. The objectives of this study are to design a guide RNA (gRNA), to insert the designed gRNA into the pGuide-it-Zs-Green1 vector, to quantify the purified CYP3A4-KO plasmid vector by using NanoDrop® and to transfect the constructed vector in a hepatic cell line. The methodology involved the selection of the gRNA by using the online gene editing tool, Synthego (https://design.synthego.com), annealing oligos of the gRNA for CYP3A4, cloning gRNA into a plasmid vector, isolation, and purification of the CYP3A4-KO plasmid vector. The construction of the CRISPR-Cas9 targeting vector in this study was successfully achieved and promising since the selected gRNA for CYP3A4 gene which is 5’-ATAAATCCCACTGGACCAAA-3’ and located in exon 5 was correctly ligated after the confirmed with sequencing reaction and cloned it into a plasmid vector. The yield of pCYP3A4-KO plasmid DNA was a good candidate for transfection. Keywords: Cytochrome P450; CYP3A4; CRISPR- Cas9; gRNA; Plasmid vector 1. Introduction Cytochrome P450 (CYP) enzymes are membrane-bound haemoproteins and are known to be the heme-thiolate proteins family [1,2]. CYP450 enzymes mainly reside in the liver and circulate throughout the body. CYP450 are monooxygenases that catalyse many reactions such as drug metabolism, and synthesis of cholesterols, steroids, and lipids [3,4]. Drug metabolism is crucial as it * Corresponding author. E-mail address: ahmadazani@uitm.edu.my https://doi.org/10.37934/araset.43.2.178188