Extended Data Fig. 10: Additional characterization of in vivo liver editing with PASTE. | Nature Biotechnology

Extended Data Fig. 10: Additional characterization of in vivo liver editing with PASTE.

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

Extended Data Fig. 10

a) PASTE integration using delivery of circular mRNA with synthetic guides and either AdV or plasmid cargo. Data are mean (n = 3) ± s.e.m. b) PASTE integration of GFP at the ACTB locus with dose titration of PASTE components and GFP cargo delivered as AdV in HepG2 cells. Data are mean (n = 3) ± s.e.m. c) Evaluation of a 3-primer NGS assay for measuring integration efficiency, akin to junctional readouts by ddPCR. Using amplicon standards mixed at predefined ratios (x-axis), we can ascertain the accuracy of the measured editing (y-axis) by NGS. d) Analysis of primary human hepatocyte (PXB-cells®) EGFP integration at the ACTB locus using adenoviral delivery for PASTEv1 and guides and AAV for the EGFP template. Viral doses are as indicated. Shown is mean ± s.e.m with n = 2. e) Analysis of all liver editing outcomes for adenoviral EGFP template integration at the ACTB locus using PASTE in vivo. f) Analysis of attB site insertion efficiency at the ACTB locus using PASTE in vivo. Data are mean (n = 8). g) Analysis of adenoviral EGFP template integration efficiency into available attB sites at the ACTB locus using PASTE in vivo. Data are mean (n = 8). h) Analysis of indel frequency at the ACTB locus using PASTE in vivo. Data are mean (n = 8). i) Analysis of attB-site associated indels during in vivo integration with PASTE via alignment of representative reads to the ACTB locus containing the desired attB site.

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