Target protein expression in T conv cells (top) and T reg cells (bottom) ( n = 2 donors) with CRISPRi targeting of the CTLA4 TSS (left, n = 3 sgRNA species for donor 2 T conv samples, n = 5 sgRNA species for all other samples) or CRISPR knockout of CTLA4 downstream of the TSS (right, n = 1 sgRNA for the donor 1 T conv CD28 sample, n = 2 sgRNA species for all other samples). Plotted are median fluorescence intensity values normalized to those of nontargeting (CRISPRi, n = 6 sgRNA species for donor 2 T conv samples, n = 7 sgRNA species for all other samples) or AAVS1 -knockout ( n = 6 sgRNA species for all samples) controls for each target gene (fill color). Mean values were compared to those of the control group using two-sided Students t -test with Holm correction (T conv CRISPRi adjusted * P = 0.047, *** P = 1.2 x 10 −10 , *** P = 3.9 x 10 −3 ; T reg CRISPRi *** P = 8.2 x 10 −5 , *** P = 4.9 x 10 −5 , *** P = 8.1 x 10 −5 ; T conv knockout P = 0.73, *** P = 1.8 x 10 −6 , P = 0.98; T reg knockout P = 1, *** P = 1.2 x 10 −17 , 1). For all panels, * P < 0.05, ** P < 0.01, *** P < 0.005; NS, not significant. Box plots indicate the sample median (central line), first and third quartiles (box) and 1.5x interquartile range (whiskers).
