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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. [ABSTRACT FROM AUTHOR]

Background: Allogeneic stem cells are the most potent sources for replacing cell, tissue, and organ malfunctions. The clinical use of these stem cells has been limited due to the risk of immune system rejection due to the incompatibility of human leukocyte (HLA) antigens between donors and recipients. To overcome this limitation, we used the CRISPR/Cas9 system to eliminate the β2 microglobulin (B2M) gene, which plays a vital role in the expression of HLA class I. Results: Non-viral transfer of two gRNAs targeting the first exon and intron in the B2M gene results in large deletions in the target region. In addition, the results of this study showed that 11.11% and 22.22% of cells received genomic changes as homozygous and heterozygous, respectively. Conclusion: In conclusion, we have shown that the dual guide RNA strategy is a simple and efficient method for modifying genes. As a result, these cells can be proposed as universal cells that are not detectable in the cell therapy system and transplantation by the receptor immune system. [ABSTRACT FROM AUTHOR]

Objective(s): Duchene muscular dystrophy (DMD) is a progressive neuromuscular disease caused by mutations in the DMD gene, resulting in the absence of dystrophin expression leading to membrane fragility and myofibril necrosis in the muscle cells. Because of progressive weakness in the skeletal and cardiac muscles, premature death is inevitable. There is no curative treatment available for DMD. In recent years, advances in genetic engineering tools have made it possible to manipulate gene sequences and accurately modify disease-causing mutations. CRISPR/Cas9 technology is a promising tool for gene editing because of its ability to induce double-strand breaks in the DNA. Materials and Methods: In this study for the exon-skipping approach, we designed a new pair of guide RNAs (gRNA) to induce large deletion of exons 48 to 53 in the DMD gene in the human skeletal muscle cell line (HSkMC), in order to correct the frame of the gene. Results: Data showed successful editing of DMD gene by deletion of exons 48 to 53 and correction of the reading frame in edited cells. Despite a large deletion in the edited DMD gene, the data of realtime PCR, immune florescent staining demonstrated successful expression of truncated dystrophin in edited cells. Conclusion: This study demonstrated that the removal of exons 48-53 by the CRISPR / Cas9 system did not alter the expression of the DMD gene due to the preservation of the reading frame of the gene. [ABSTRACT FROM AUTHOR]

Background: The treatment of many cancers and genetic diseases relies on novel engraftment approaches such as cell therapy and hematopoietic stem cell transplantation (HSCT). However, these methods are hindered by the alloreactive immune responses triggered by incompatible human leukocyte antigen (HLA) molecules. A successful HSCT procedure requires the eradication of donor and recipient HLA alloimmunization. Eliminating HLA-A gene expression using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 nuclease (CRISPR/Cas9) could be a great approach to increase the possibility of a successful HSCT through extending pool of unrelated donors. Results: Our dual gRNA approach introduced a large deletion in the HLA-A gene. Among 22 single-cloned cells, two clones (9.09%) and 11 clones (50%) received homozygous and heterozygous large deletions, respectively. Finally, the real-time PCR results also revealed that HLA-A gene expression was diminished significantly. Conclusion: The results suggested that CRISPR/Cas9 could be used as an efficient technique to introduce HLA-A gene knockout; thus, it can considerably lessen the burden of finding a fully matched donor by lowering the alleles required for a successful HSCT. [ABSTRACT FROM AUTHOR]