CRISPR-Cpf۱ mediated targeting of TRAC locus in T cell line

Genetically engineered T cell therapy is employed for various types of cancer and it has shown considerable therapeutic potential, both for solid tumors and blood cancers. However, endogenous TCRs at the surface of the engineered T cells, have faced these treatments with the challenge of GVHD (graft versus host disease) for allogeneic approach, where these endogenous TCRs reacts against the recipient tissue. Whereas, TCRαβ at the surface of engineered T cells causes this allogeneic reaction, prevent the expression of functional TCRαβ at the surface of engineered T cells can solve this challenge. In this study, we have assessed the application of CRISPER/Cpf۱ gene editing system to knock out TCRα chain in a Jurkat T cell line. To target the TRAC locus that encodes the constant region of TCRα, three crRNAs was designed and constructed. The efficiency of this gene editing system was assessed in two levels, genome sequence and protein expression, respectively, was examined by using DNA sequence analysis and flowcytometry. The results showed that two of three designed crRNAs against TRAC locus, introduced indel mutations in the constant region of TCRα and prevented the functional TCR expression at cell surface. These findings show that CRISPER/Cpf۱ is appropriate gene editing system in order to remove endogenous TCR from the surface of Jurkat T cell line. Using two crRNAs designed in this study, CRISPER/Cpf۱ gene editing system can be applied to remove endogenous TCR from the surface of primary T cells for manufacture of allogeneic engineered T cell therapy.

کلیدواژه ها:

CRISPER/Cpf۱ ، TRAC locus knockout ، endogenous TCR depletion