Shiraz University of Medical Sciences Int Cardio Res J 2251-9149 16 4 2023 Jan 31 Mahdieh Talebzadeh Vahid Razban Mahintaj Dara Hossein Jafari Khamirani Maryam Ranjbar Marjan Nourigorji Farid Torabizadeh Mehdi Dianatpour Research Article 2023 05 14 2023 05 14 2023 Jan 31 2022 12 31 : Background: Cardiovascular diseases, with an estimated 18.6 million deaths per year,
are the leading cause of death worldwide. One of the major risk factors is elevated blood
low-density lipoprotein cholesterol (LDL-C) secondary to multiple environmental and
genetic factors. Genes involved in LDL-C metabolism are the targets of the most common
treatment options. Advanced molecular techniques could pave the way for identifying
novel targets in dyslipidemia therapies. The LIM domain and actin-binding 1 (LIMA1)
gene binds to the NPC1L1 protein and facilitates its more efficient recycling to the plasma
membrane. Inhibition of LIMA1 could disrupt cellular cholesterol hemostasis with a
probable decrease in blood LDL-C levels.
Objectives: The present study was designed to knock out exon 2 of the LIMA1 gene using
lentiviruses as an in vitro model for reducing cholesterol absorption.
Methods: A CRISPR/Cas9 system with dual guide RNAs (gRNAs) was designed to
completely excise exon 2 of LIMA1. Two gRNAs (gRNA1 and gRNA2) were cloned in
the LentiCRISPR v2 vector. LentiCRISPR viruses were produced in the HEK293T cell
line to encode the CRISPR/Cas9 complex structure. HepG2 cell lines were transduced
with two different LentiCRISPR viruses simultaneously.
Results: Exon 2 deletion was detected by PCR, gel electrophoresis, and subsequent
Sanger sequencing of the PCR product. Exon 2 deletion caused a frameshift mutation,
and the subsequent production of nonfunctional transcripts led to gene knockout. The
dual gRNA CRISPR/Cas9 system could be used in gene editing setups.
Conclusions: The in vitro knockout model of LIMA1 could be considered as preliminary
work to study the role and mechanism of action of the LIMA1 protein, along with its
potential as a target for hypercholesterolemia therapy. https://brieflands.com/articles/ircrj-131422.html No Link 103281 11695 104998 1510 104999