Extended Data Fig. 8: Therapeutic applications of PASTE and further characterization of integrases. | Nature Biotechnology

Extended Data Fig. 8: Therapeutic applications of PASTE and further characterization of integrases.

From: Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases

Extended Data Fig. 8

a) Schematic of protein production assay for PASTE-integrated transgene. SERPINA1 and CPS1 transgenes are tagged with HIBIT luciferase for readout with both ddPCR and luminescence. b) Integration efficiency of SERPINA1 and CPS1 transgenes in HEK293FT cells at the ACTB locus. c) Integration efficiency of SERPINA1 and CPS1 transgenes in HepG2 cells at the ACTB locus. d) Intracellular levels of SERPINA1-HIBIT and CPS1-HIBIT in HepG2 cells. e) Secreted levels of SERPINA1-HIBIT and CPS1-HIBIT in HepG2 cells. f) Integration of SERPINA1 and CPS1 genes that are HIBIT tagged as measured by a protein expression luciferase assay. g) Integration of SERPINA1 and CPS1 genes that are HIBIT tagged as measured by a protein expression luciferase assay normalized to a standardized HIBIT ladder, enabling accurate quantification of protein levels. h) PASTE integration activity with most active integrases compared to BxbINT. i) Characterization of integrase activity on truncated attachment sites using integrase reporters in HEK293FT cells. j) PASTE integration activity with computationally selected integrases with shorter attB sites. Data are mean (n = 3) ± s.e.m.

Back to article page