Genome engineering is a novel and powerful tool for biotechnological applications. the development of the clustered regularly interspaced short palindromic repeats (crispr)-cas9 system has revolutionized the field of gene editing. the purpose of this study was to knock out trbc (tcr β constant regions) genes. trbc1 and trbc2 genes encode the constant part of the β chain of t cell receptors (tcr). crispr/cas9-mediated tcr knockout has therapeutic implications where donor-derived t cells are induced to achieve anti-tumor effects. the endogenous αβtcr on infused allogeneic t cells may recognize major and minor histocompatibility antigens of the recipient, leading to graft-versus-host-disease (gvhd).
Using available online tools, we designed a grna targeting a sequence common to both trbc1 and trbc2 genes. then grna oligo was cloned into crispr/cas9 plasmid using golden gate assembly cloning strategy.
A grna was designed and cloned to cleave the consensus coding sequence of human trbc genes. proper insertion of the grna was then verified by sanger sequencing. we plan to transfect the verified clones into appropriate cell lines and further analyze the cells at genomic and protein levels.
Here we present a method to circumvent gvhd in allogeneic t-cell-based therapies. our method exploits crisper/cas9 system to knock out trbc genes, leading to the elimination of tcrs; which in turn has been shown to prevent gvhd.