Extended Data Fig. 1: In vivo human CAR-T cell screening results are comparable across multiple healthy human donors.

a. Construct designs for the 4-1BB BCMA CAR (pCAR) and double-guide cassette (pGuide) containing the Mario sgRNA library (variable sgRNA). b. Timeline of the MM.1S stress model with 21 day tumor engraftment and 2E6 CAR treatment. Tumor growth was tracked by BLI. Data presented as mean +/– SEM. n = 3 mice (tumor only, Mario library (no CAR)) and n = 5 (CAR-T, Mario-CAR-T) per group from one healthy donor (ND202). Statistical significance amongst groups at day 35 as measured by two-way ANOVA with Tukey’s multiple comparison. c. CD3 expression after day -5 CD3 negative enrichment (ND216, ND99, ND106). d. Representative Mario-CAR-T cell staining for CD34 indicating CAR transduced cells and NGFR for sgRNA library transduced cells from ND216. e. Replicate autocorrelation analysis scatter plots. Pearson’s correlations are calculated for the library distribution of one animal versus any other animal, two averaged animals versus any other two, and so on. The mean of all possible combinations is plotted. f. Quantification of replicate autocorrelation analysis. Pearson’s correlations are calculated for the library distribution of one animal versus any other animal, two averaged animals versus any other two, and so on. The mean of all possible combinations is plotted. g. z-scored abundance histograms of gene targeting or intergenic control sgRNAs across screening time points and conditions. h. Donor Pearson correlations across screening time points and conditions. ns: non significant.