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

 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. 

 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. 

 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. 

 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. 

 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.