LIMA1 Gene Knockout by CRISPR/Cas9 System Using Lentiviruses as an in Vitro Model for Reducing Cholesterol Absorption

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 bloodlow-density lipoprotein cholesterol (ldl-c) secondary to multiple environmental andgenetic factors. genes involved in ldl-c metabolism are the targets of the most commontreatment options. advanced molecular techniques could pave the way for identifyingnovel 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 plasmamembrane. inhibition of lima1 could disrupt cellular cholesterol hemostasis with aprobable decrease in blood ldl-c levels.objectives: the present study was designed to knock out exon 2 of the lima1 gene usinglentiviruses as an in vitro model for reducing cholesterol absorption.methods: a crispr/cas9 system with dual guide rnas (grnas) was designed tocompletely excise exon 2 of lima1. two grnas (grna1 and grna2) were cloned inthe lenticrispr v2 vector. lenticrispr viruses were produced in the hek293t cellline to encode the crispr/cas9 complex structure. hepg2 cell lines were transducedwith two different lenticrispr viruses simultaneously.results: exon 2 deletion was detected by pcr, gel electrophoresis, and subsequentsanger sequencing of the pcr product. exon 2 deletion caused a frameshift mutation,and the subsequent production of nonfunctional transcripts led to gene knockout. thedual grna crispr/cas9 system could be used in gene editing setups.conclusions: the in vitro knockout model of lima1 could be considered as preliminarywork to study the role and mechanism of action of the lima1 protein, along with itspotential as a target for hypercholesterolemia therapy.