ED Fig. 3. The ABI domain increases Cas9 loading into EVs and Cas9-ABI retains function.

a, Expression of Cas9 fused to either the ABI or PYL domain in transiently transfected HEK293FT cells. 2 μg cell lysate was loaded per lane. Expected band sizes: ~160, 183, and 195 kDa (arrows). b, Cartoon illustrating the Cas9 reporter construct. Successful editing by Cas9 results in the deletion of a stop codon and (in some random fraction of cases) a repair-mediated frame shift induces express dTomato. c, Absence of an NLS or presence of the ABI domain does not meaningfully reduce Cas9 editing efficiency in transiently transfected Jurkat T cells. Cells were analyzed by flow cytometry 3 d post-transfection. Experiments were performed in biological triplicate, and error bars indicate standard error of the mean. Statistical tests comprise two-tailed Student’s t-tests using the Benjamini-Hochberg method to reduce the false discovery rate. (*p < 0.05, **p < 0.01, ***p < 0.001). Exact p-values are reported in Supplementary Table 1. Samples with high cellular autofluorescence were excluded from analysis. d, Full blot of Cas9 EV active loading data presented in Fig. 3e. e, Cellular expression of Cas9 with and without the ABI domain or an NLS. 2 μg cell lysate was loaded per lane.