CRISPR/Cas9-mediated Genome Editing

Adoptive immunotherapy with genetically-engineered T-cells, specially CAR-T cell therapy, has shown promise in clinical trials. However, this approach is limited by the requirement of generating autologous, patient-derived, T-cells, which is costly, labor-intensive, and time-consuming. To create CAR-T therapy more attainable, a methodology which allow the generation of allogeneic universal T-cells would be highly recommended. When allogeneic CAR T cell infusion is considered, host versus graft and graft versus host reactions must be avoided to elicit successful antitumor activity. In this project, we exploit the CRISPR/Cas9-mediated editing system to develop a process for manufacturing of T cells deficient in expression of some alloreactive antigens.gRNAs design using the available online tools, cloning of the gRNAs oligo inserts into the pSpCas9(BB)-2A-GFP and pSpCas9(BB)-2A-Puro plasmids by Golden Gate Assembly cloning strategy, Sequence validation of CRISPR plasmids, Transfection of sequence verified plasmids into HEK293 Cell line (functional validation), Selection of transfected Cells, Screening of CRISPR/Cas9-Mediated Deletions.gRNAs were designed and cloned to recognize and cleave the coding sequences of human TRAC and CD52 genes. Two or three colonies were picked to check for the correct insertion of gRNAs. The sequence of each colony was verified by sequencing to check the insertion of guide sequence between the U6 promoter and the remainder of the gRNA scaffold. The sequence verified clones were selected to transfect the HEK293 cell line to validate the efficiency of gRNAs. Transfected cells were selected by FACS or antibiotic selection to validate the functional efficiency at genomic level.

Keywords:

Adoptive Immunotherapy , Gene editing , Universal T Cell , CRISPR/Cas9