The Construction of CRISPR-Cas9 System Targeting Vector for CYP3A4 Gene in Hepatic Cell Lines

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 aroun...

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Published in:Journal of Advanced Research in Applied Sciences and Engineering Technology
Main Author: Hatta F.H.M.; Aziz F.N.A.; Rehman S.-U.; Othman A.A.
Format: Article
Language:English
Published: Semarak Ilmu Publishing 2025
Online Access:https://www.scopus.com/inward/record.uri?eid=2-s2.0-85192187841&doi=10.37934%2faraset.43.2.178188&partnerID=40&md5=c687df7c0639839dd4b59f88805137f3
id 2-s2.0-85192187841
spelling 2-s2.0-85192187841
Hatta F.H.M.; Aziz F.N.A.; Rehman S.-U.; Othman A.A.
The Construction of CRISPR-Cas9 System Targeting Vector for CYP3A4 Gene in Hepatic Cell Lines
2025
Journal of Advanced Research in Applied Sciences and Engineering Technology
43
2
10.37934/araset.43.2.178188
https://www.scopus.com/inward/record.uri?eid=2-s2.0-85192187841&doi=10.37934%2faraset.43.2.178188&partnerID=40&md5=c687df7c0639839dd4b59f88805137f3
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. © 2025, Semarak Ilmu Publishing. All rights reserved.
Semarak Ilmu Publishing
24621943
English
Article
All Open Access; Hybrid Gold Open Access
author Hatta F.H.M.; Aziz F.N.A.; Rehman S.-U.; Othman A.A.
spellingShingle Hatta F.H.M.; Aziz F.N.A.; Rehman S.-U.; Othman A.A.
The Construction of CRISPR-Cas9 System Targeting Vector for CYP3A4 Gene in Hepatic Cell Lines
author_facet Hatta F.H.M.; Aziz F.N.A.; Rehman S.-U.; Othman A.A.
author_sort Hatta F.H.M.; Aziz F.N.A.; Rehman S.-U.; Othman A.A.
title The Construction of CRISPR-Cas9 System Targeting Vector for CYP3A4 Gene in Hepatic Cell Lines
title_short The Construction of CRISPR-Cas9 System Targeting Vector for CYP3A4 Gene in Hepatic Cell Lines
title_full The Construction of CRISPR-Cas9 System Targeting Vector for CYP3A4 Gene in Hepatic Cell Lines
title_fullStr The Construction of CRISPR-Cas9 System Targeting Vector for CYP3A4 Gene in Hepatic Cell Lines
title_full_unstemmed The Construction of CRISPR-Cas9 System Targeting Vector for CYP3A4 Gene in Hepatic Cell Lines
title_sort The Construction of CRISPR-Cas9 System Targeting Vector for CYP3A4 Gene in Hepatic Cell Lines
publishDate 2025
container_title Journal of Advanced Research in Applied Sciences and Engineering Technology
container_volume 43
container_issue 2
doi_str_mv 10.37934/araset.43.2.178188
url https://www.scopus.com/inward/record.uri?eid=2-s2.0-85192187841&doi=10.37934%2faraset.43.2.178188&partnerID=40&md5=c687df7c0639839dd4b59f88805137f3
description 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. © 2025, Semarak Ilmu Publishing. All rights reserved.
publisher Semarak Ilmu Publishing
issn 24621943
language English
format Article
accesstype All Open Access; Hybrid Gold Open Access
record_format scopus
collection Scopus
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