Fig. 4. Multiplex CRISPR-cell engineering enhances cartilage deposition.

(A) Expression cassettes for the CRISPRa system and upregulation of Col2A1, ACAN, and ZNF865. After 21-days, (B) gross pellet morphology showed increases in overall tissue deposition (scalebar = 1 mm) and increased staining for (C) alcian blue indicating increased GAG deposition (scalebar = 100 μm). Biochemical analysis showed significant increases in (D) μg GAG/pellet, (E) % GAG retained in each pellet, (F) μg collagen/pellet, and (G) % collagen retained in each pellet for ACAN/Col2a1-ZNF865 edited hASCs compared to ACAN/Col2a1-NTC and naïve hASCs without GFs (n = 5–10, *=p < 0.05). NTC is nontarget control, GFs is growth factors.