This study aimed to evaluate the efficiency of CRISPR/Cas9-mediated genome editing with dual guide RNA as a simple method for genetic manipulation to produce null HLA class 1 donor cells that could potentially address graft-related problems. Due to the B2M subunit's structural importance, the reduced B2M expression has led to the incorrect formation and inactivation of HLA type 1 [21,22,23]. As a result of the eradication of cell surface expression of polymorphic HLA class I molecules (HLA-A,-B,-C), allograft cells have the chance to evade the host immune system and are not recognized by the CD8 + T cells [24, 25]. The CRISPR/Cas9 system allows us to delete the B2M gene and cut a relatively large portion of DNA by simply inserting two guide RNAs into the cell (Fig. 3). In this study, a third portion—12 out of 36 clones—of the cells was mutated by dual Cas9 cleavage. Notably, the creation of relatively large deletion within the target region would enable us to employ a fast and straightforward method such as PCR for genotyping the modified genes. Our data and findings from other studies clearly revealed the benefits of utilizing the dual gRNAs approach in gene knockout. It was already shown that this efficient strategy could be employed to study repetitive sequences in which genetic manipulations via single-guide RNA results in increased off-target effects. In addition, the successful applicability of dual gRNA CRISPR/Cas9 technology in perturbing non-coding regions, including silencers, enhancers, and long non-coding RNAs, has been reported [7, 26]. According to previous studies, small indel mutations are more likely to lead to the loss of function in non-coding regions using a gRNA-based CRISPR/Cas9 system [7, 27]. In addition, the use of single gRNAs in CRISPR/Cas9 systems can lead to the production of various small indels in target sequences that require laborious T7 endonuclease 1 (T7E1) assay to detect Cas9 activity [28]. Furthermore, employing a single gRNA method cannot efficiently abate b2m in specific cell types such as T cells [7].
Hong et al. simultaneously produced HLA class I cells by targeting HLA-A/B/C genes using six gRNAs. The efficiency of the method used in this study was less than the method used in our study. According to the results of their study, the simultaneous transfer of six gRNAs to the cell reduced the survival of target cells. Also, selecting cells simultaneously under the influence of six gRNAs would be technically demanding [29]. In other studies conducted by Xu et al. and Jang et al., the HLA-A and/or HLA-B was successfully ablated in inducible pluripotent stem cells (iPSC) instead of complete disruption of HLA class I [25, 30]. Although they could attain cells with less immunogenicity, there will still be HLA disparity for alleles retaining between donor and recipient, which should be assessed.
Another alternative way to provide an optimal source of donor cells is to remove HLA class II under certain conditions. Although targeting the CIITA gene can lead to an expansion of the immune system, elimination of HLA class II due to impaired maturation of CD4 + T cells can lead to lymphopenia [25, 31].
Although many in vivo studies on animal models are solely concentrated on T cells, some studies recognized B cells in the transplanted organ for their ability to develop into long-lived plasma cells that produce high-affinity alloantibodies. Also, in addition to T cells, B cells are recognized to have a role in transplant rejection by donor-specific antibody production and otherwise may lead to tolerance when acting as an antigen-presenting cell [32].
The generation of universal cells using the dual gRNAs approach can resolve the issues such as the shortage of suitable donors and graft rejections. Moreover, these HLA class I null-cells can serve as a source of artificial antigen-presenting cells to produce cytotoxic T cells. However, this area has many limitations, including the identification and destruction of cells by natural killer cells (NK cells). To solve this problem, overexpression of non-classical HLA type 1 protein has been suggested to reduce the emerging status of NK cells [31]. Tumor formation due to HLA class I deficiency after transplantation is another problem in this field. The artificial introduction of suicide or apoptosis genes has been proposed to prevent tumor formation. In addition, it has been suggested that artificially engineered Caspase 9-induced artificial apoptosis has shown promising results because it is more effective, safer, and more immunogenic than the previous approach [10, 33].