Fig. 2
Gene editing in intact pre-implantation embryos transduced with rAAV vectors. a Schematic representation of the strategy to transduce C57BL/6NJ zygotes with rAAV vectors designed to target the Tyrosinase (Tyr) locus. Zygotes were placed in KSOM containing rAAV6-Cas9 and rAAV6-sgTyr vectors, rinsed, cultured at 37 °C for 3 days, and analyzed for Tyr gene editing at compacted morula or blastocyst stages. Alternatively, they were cultured overnight and transferred at the 2-cell stage into the oviducts of E0.5 pseudopregnant females. Transferred embryos were assessed for eye pigmentation at E16.5, or allowed to develop to birth and assessed for eye and coat color pigmentation. b Stacked histogram showing the percentage distribution of indel-type frequencies among four rAAV-dosage groups . Alterations indicate base replacements; large deletions are defined as removal of >20 bases and compound mutations are combinations of insertions, deletions, and/or alterations. c Analysis of eye pigmentation in E16.5 embryos transduced with rAAV6-Cas9 only (left panel) and both rAAV6-Cas9 and rAAV6-sgTyr (right panel). Arrow in the right panel indicates the location of the eye in a transduced albino embryo. d Albino litter generated after transduction of C57BL/6NJ zygotes with CRISPR-Cas9 rAAV vectors at 6 × 109 GC dose. Shaved area on female indicates site of embryo transfer surgery. e A representative litter obtained after transduction of C57BL/6NJ zygotes with CRISPR-Cas9 rAAV vectors at 6 × 108 GC dose. Three out of five pups are albino and two are mosaic as revealed by the variegated coat color pattern. f Schematic representation of the strategy to test germline transmission of CRISPR-Cas9-induced alleles of Tyr. Tyr-edited albino mice were mated with albino CD-1 (Tyrc/c) animals and the offspring were assessed for the presence of albino coat color. g Litter derived from Tyr-edited albino male crossed with a CD-1 female. All pups are albino indicating germline transmission
