FIGURE 3.
Com and com interaction enriched Cas9 RNPs into EVs. [A. Enriching SaCas9 RNPs in EVs. Com‐CD63‐Com, SaCas9 and IL2RG sgRNA with and without com modification were co‐expressed in 5 × 106 HEK293T cells, EVs were collected for 48 h. One fifth EVs were analyzed in Western blotting. Numbers under protein bands were estimated protein mass (ng) based on protein standards. B. Enriching SpCas9 and ABE RNPs into EVs. IL2RG and Site 5 were targeted for SpCas9 and ABE respectively. Experimental conditions were similar as in A. C. Com and com dependent enrichment of sgRNA into EVs. SaCas9 and IL2RG‐targeting sgRNA were packaged in EVs (n = 3). sgRNA was unmodified (com−) or with a com replacing the Tetra loop (com+). In EVs without Com‐CD63‐Com, CD63 was overexpressed instead. RNAs were extracted from EVs and the sgRNA was detected by RT‐qPCR using primers Scid‐g2F and sgRNA‐R3 (See Table S2). ***, P < 0.0001 when compared with all other conditions (Tukey posttests following ANOVA)]