HLA-A Gene Knockout Using Dual gRNA by CRISPR-Cas9 System

Background and Aim: Nowadays, the treatment of many cancers andgenetic diseases is focused on cell therapy and long-term allogenicengraftment, which the most important is HLA molecules. Wedemonstrated that HLA-A molecule on the HEK293T cell line couldbe eliminated by CRISPR-Cas9, which would make the process oftransplantation easier.Methods: Two gRNAs with the highest efficiency and the lowest offtargetwere designed by existing databases. Then, these gRNAs wereseparately inserted in the px458 vector, transformed and cloned in E.coli. The insertion of gRNAs into the vector validated by both PCR andSanger sequencing, and then transfected into the HEK293T by lipofection2000. Green fluorescent transfected cells sorted by FACS, and singlecell dilution performed. Afterward, the DNA’s from every single cloneextracted and then both PCR and Sanger sequencing approved excisedregion by CRISPR-Cas9.Results: After selecting the HEK293T cell as a model, HLA typing wasperformed and the allele was determined. The design of gRNAs wascarried out according to the type of cell allele and for exons 2 and 3 ofHLA-A2. After co-transfection of the plasmids, doing FACS and single celldilution, the proportion of HLA-A gene-disrupted cells was 9.09%. At2–3 weeks after sorting, 22 clones among a total of 192 wells (11.45%)were cultured and analyzed for HLA-A gene mutations.Conclusion: The studies showed that removing both alleles of theHLA-A gene not only protects the cell against immune responses butalso significantly increases the chance of finding an unrelated donor byreducing the number of alleles required for allogeneic transplantation. Ithas also been shown that the CRISPR-Cas9 system is an efficient and safetechnique to perform gene knockout.

کلیدواژه ها:

HLA-A2 antigen ، Gene Knockout Techniques ، Transplantation ، CRISPR-CAS Systems