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. 2023 May 4;42(2):253–264. doi: 10.1038/s41587-023-01758-z

Fig. 4. Development of a dual-AAV system for efficient in vivo prime editing.

Fig. 4

The RNaseH domain of PE3 is not essential in the liver, heart, or brain. a, C57BL/6 mice were RO injected with 1.25 × 1012 vg total of v2em PE3-AAV9 (5 × 1011 vg each N-terminal and C-terminal AAVs plus 2.5 × 1011 vg epegRNA/sgRNA AAV) encoding PE3max with its native full-length RT or PE3max with one of two truncated ΔRNaseH RTs to install a Dnmt1 +1 C-to-G edit. Three weeks after injection, liver, heart, and muscle tissues were harvested and analyzed by HTS. Dots represent individual mice, and error bars represent mean ± s.e.m. of n = 1–3 mice; each condition includes both male and female mice. b, C57BL/6 pups were injected on P0 by ICV injection of a total of 5.7 × 1010 vg total of v2em PE3-AAV (2.3 × 1010 vg each N-terminal and C-terminal AAVs plus 1.1 × 1010 vg epegRNA/sgRNA AAV) encoding PE3max with full-length RT or PE3max with a truncated ΔRNaseH RT to install the Dnmt1 +2 G-to-C edit. Three weeks after injection, mice were harvested; cortex was dissected; and nuclei were isolated and sorted by FACS. The bulk population (all nuclei) and a GFP+ subpopulation were lysed, and gDNA was analyzed by HTS. Dots represent individual mice, and error bars represent mean ± s.e.m. of n = 3–4 mice; each condition includes both male and female mice. c, Schematic of v3em PE3max AAV. d, C57BL/6 mice were RO injected with either 1.25 × 1012 vg total of v2em PE3 ΔRNaseH AAV9 (5 × 1011 vg each N-terminal and C-terminal AAVs plus 2.5 × 1011 vg epegRNA/sgRNA AAV) or 1 × 1012 vg total of v3em PE3-AAV9 (5 × 1011 vg each N-terminal and C-terminal AAVs), both installing the Dnmt1 +1 C-to-G edit. Three weeks after injection, liver, heart, and muscle tissues were harvested and analyzed by HTS. Dots represent individual mice, and error bars represent mean ± s.e.m. of n = 3–5 mice; each condition includes both male and female mice. e, Prime editing efficiency in bulk liver of v1em or v3em PE3-AAV9 after a single injection of AAV at two different doses. C57BL/6 mice were RO injected with 1 × 1011 vg or 1 × 1012 vg total of either v1em or v3em PE3-AAV9 containing an epegRNA encoding the Dnmt1 +2 G-to-C edit. Three weeks after injection, liver tissue was harvested and analyzed by HTS. Dots represent individual mice, and error bars represent mean ± s.e.m. of n = 4 mice; each condition includes both male and female mice.