(A) CRISPR/Cas9 RNP-mediated genome editing at the TCRA and TCRB genomic loci in primary human T cells. Location of gRNA sites targeting the TCRA 3’-UTR, to generate TCRA ΔPAR cells is highlighted in red. In TCRA, gRNA1 and gRNA2 target hg38 genomic locations chr14:22,551,700 and chr14:22,552,073, respectively. Location of gRNA sites targeting the TCRB 3’-UTR, to generate TCRB ΔPAR cells is highlighted in blue. In TCRB, gRNA1 and gRNA2 target hg38 genomic locations chr7:142,802,561 and chr7:142,802,695, respectively. (B) Top gel: Analysis of TCRA 3’-UTR PAR-CLIP site deletion efficiency. Total genomic DNA extracted from WT, TCRA ΔPAR, TCRB ΔPAR (here serving as a negative control), scrambled gRNA nucleofected (S) and non-nucleofected (N) cells was analyzed by PCR to measure the editing efficiency. TCRA ΔPAR cells produced a 1283 bp PCR product compared to 1676 bp in S, N or TCRB ΔPAR cells (See panel A). Bottom gel: Analysis of TCRB 3’-UTR PAR-CLIP site deletion efficiency. Total genomic DNA extracted from WT, TCRA ΔPAR (here serving as a negative control), TCRB ΔPAR, scrambled gRNA nucleofected (S) and non-nucleofected (N) cells were analyzed by PCR to measure the editing efficiency. TCRB ΔPAR cells produced a 1022 bp PCR product compared to 1190 bp in S, N or TCRA ΔPAR cells (See panel A). The percentage of alleles with PAR-CLIP site deletion, quantified by imageJ. (C) Representative immunofluorescence images used to count TCR clusters in WT, TCRA ΔPAR and TCRB ΔPAR primary human T cells. The white arrows indicate the cells that were scored as having TCR clusters. (D) Percentage of cells expressing TCR on the cell surface of activated T cells measured by flow cytometric analysis. TCR expressing cells in the cell populations tested: N, Non-nucleofected cells; SC, Scrambled gRNA; 1gA, TCRA gRNA 1; 2gA, TCRA gRNA 2; 1 + 2 gA, TCRA gRNA 1 + 2 (i.e. TCRA ΔPAR); 1 gB, TCRB gRNA 1; 2 gB, TCRB gRNA 2; 1 + 2 gB, TCRB gRNA 1 + 2 (i.e. TCRB ΔPAR); ΔTCR, TCR gRNA targeting the CDS of TCRA. (n = 3 replicates from three separate wells, with mean and standard deviation shown.) (E) Representative density plots showing the percentage of TCR on the cell surface in activated T cells. The cell lines shown are: SC, TCRA ΔPAR, TCRB ΔPAR, ΔTCR (negative control). In panels D and E, representative results from one of two donors are shown. (F) Gating strategy for flow cytometric analysis of CD69 and CD25 levels. Primary human T cells were gated to isolate lymphocytes, followed by isolation of single cells, then live cells. Next, cells were gated to separate CD4+ and CD8+ cells expressing T cell activation markers CD69 and CD25. The gates for CD69+ and CD25+ cells were set based on CD69 and CD25 levels in the ΔTCR T cells. Shown is the workflow of the FACS gating, with an example of T cells activated with anti-CD3/anti-CD28 antibodies for 8 hr.