Edit-R Synthetic crRNA T-cell Receptor Targets

Validated Edit-R guide RNAs targeting T Cell Receptor genes

T Cell Receptor genes are made up of variable and constant regions that undergo rearrangement at the genomic level during T cell development to create a vast repertoire of receptors. These receptors are critical for the recognition of foreign antigens and the subsequent immune response. The complexity of these genomic rearrangements makes CRISPR guide RNA design challenging in these regions. We have used our decades of experience with algorithm development and RNA design to overcome these design challenges to successfully create and validate guide RNAs that target and efficiently knockout the T cell receptors.

We validated the guides showing a >90% TCR knockout efficiency using RNP-based editing in primary CD4+ T cells, CD8+ T cells, and induced pluripotent stem cells (iPSCs) and functionally characterized CD8+ CAR-T cells after TCR α/β knockout. Read the High-efficiency endogenous TCR knock-out and functional validation of CD8+ CAR-T cells - App Note to learn more.

TRAC (GeneID: 28755; TCRA, IMD7, TRCA, TRA, TCRA, T cell receptor alpha constant): T cell receptors recognize foreign antigen that have been processed into peptides and bound to MHC molecules at the surface of antigen presenting cells. After rearrangement of the V(variable)-D(diversity)-J(joining) segments at the DNA level, T cell receptor alpha chain constant (TRAC) region is spliced to the V-D-J segment of the T cell receptor alpha chain at the RNA level.

TRBC1/TRBC2 (GeneID: 6957; TRB, TCRB, T cell receptor beta locus/cluster): T cell receptors recognize foreign antigen that have been processed into peptides and bound to MHC molecules at the surface of antigen presenting cells. After rearrangement of the V(variable)-D(diversity)-J(joining) segments at the DNA level, T cell receptor beta chain constant (TRBC) region is spliced to the V-D-J segment of the T cell receptor beta chain at the RNA level.

TRDC (GeneID: 28256; TRDC, T cell receptor delta constant): T cell receptors recognize foreign antigen that have been processed into peptides and bound to MHC molecules at the surface of antigen presenting cells. After rearrangement of the V(variable)-D(diversity)-J(joining) segments at the DNA level, T cell receptor delta chain constant (TRDC) region is spliced to the V-D-J segment of the T cell receptor delta chain at the RNA level.

Edit-R synthetic guide RNAs targeting T cell receptor constant regions are validated for high editing efficiency while maintaining high viability in primary T cells

Primary CD4+ T cells from two donors were nucleofected with Cas9-RNP complexes loaded with TRAC single sgRNAs (donor 1 n=3, donor 2 n=2, +/-SD). 72 hours after nucleofection, functional knockdown was assessed by flow cytometry for TCR α/β receptor expression using BioLegend APC anti-human TCR α/β antibody. Cells were co-stained with BioLegend Zombie Green Viability Dye to gate on the living cell population (Zombie Green Neg) of edited cells (displayed on the right y-axis, green diamonds). Overlaid histograms from the top performing sgRNAs are pictured to the right of the bar graph in blue (top), unedited (no sgRNA) and PPIB control sgRNA – both positive controls for TCR α/β gating in red and purple, respectively (below). For a full description of the methods, see our T cell electroporation protocol.

TCR knock-out is stable in CD8+ T cells

Primary CD8+ T cells were electroporated with Cas9-RNP loaded with Edit-RTM PPIB, NTC2, TRAC or TRBC sgRNAs. 72 hours post electroporation, edited cells were assessed for TCR α/β knock-out phenotype by flow cytometry. Displayed in overlapping histograms are ZombieGreenTM Dye-negative (living) unedited CD8+ T cells unstained (black), isotype stained (gray) or CD8+ T cells that were stained with PE anti-human TCR α/β antibody (blue, green, and orange traces in histograms). For a full description of the methods, see our T cell electroporation protocol and CD8+ CAR-T application note.