Universal gRNAs Design to Increase Chance of Finding a Suitable Donor for Hematopoietic Stem Cell Transplantation UsingCRISPR/Cas9 System

Background and Aim: Human Leukocyte Antigen (HLA) molecules are acomplex system that expresses as major Histocompatibility Complex inthe human and it is one of the most polymorphic regions of the genome.These molecules have an important role in transplantation and adaptiveimmunity; therefore, mismatch of HLA alleles can lead to graft rejectionand graft-versus-host disease. We developed a strategy to address thisissue for the Iranian population using a CRISPR-Cas9 system.Methods: According to previous studies, the most common HLA-Aalleles in the Iranian population were collected. Based on this, 14common alleles were selected and the sequence of each allele wasextracted individually from https://www.ebi.ac.uk/ipd/imgt/hla/. Usingthe alignment tools (https://www.ebi.ac.uk/ipd/imgt/hla/align.html),we aligned all the alleles simultaneously. In order to design a commongRNA among the identical areas of aligned sequences, we referred tothe https://crispr.mit.edu. After analyzing gRNAs, the best gRNAs wereselected based on efficiency and off-target.Results: From https://crispr.mit.edu database, two gRNAs were pickedup. gRNA1 covers all HLA-A alleles except HLA-A1. To encompass allHLA-A alleles, another gRNA named gRNA2 was selected, which itcovers four alleles including HLA-A1.Conclusion: Despite the high level of polymorphisms in HLA genes, twouniversal gRNAs for Iranian population were designed that it can be usedfor approximately (99.1%) all Iranian racial populations. In this way, withthe new technology for gene editing, CRISPR-Cas9 can induce indel inthe HLA-A gene; consequently, the cell cannot express HLA-A moleculeon its surface. Failure to express this gene will make it much easier to finda proper donor in the population.

کلیدواژه ها:

HLA Antigens ، CRISPR-Cas systems ، gRNA ، Transplantation ، Iran ، RNA ، Guide