Fig. 2

eGFP expression following Cas9 injection. a Breeding strategy and pedigree of eGFP⁺ colonies described in this paper. Colony 347–10 is a founder derived from an injected embryo and is assumed to be a mosaic of edited eGFP⁺ cells and wild-type eGFP⁻ cells. Dotted lines indicate backcrosses. b Larvae injected with Cas9:sgRNA_847 complexes + repair template pUP613 at 144 hpf post-injection, showing mosaic expression of eGFP. An uninjected 144 hpf larvae is shown for comparison. Specks of green auto-fluorescence are common in uninjected larvae (arrowhead) but are easily distinguished from eGFP expression. c Left panel: dark-field image of colony 347–10, showing gonozooids (white asterisk). Right panel: Fluorescence micrograph of the same area of colony 347–10 showing eGFP expression. Developing oocytes (arrowheads) are intensely fluorescent, but become dim as they mature (arrows). d Darkfield (top) and fluorescence (bottom) micrograph of 24 hpf embryos from the backcross of 347–10 to 291–10, beginning to show slightly dimmer eGFP signal in some larvae (arrowhead) compared to others (arrow). e 72 hpf embryos from the same cross. eGFP⁺ (arrow) and eGFP^dim (arrowhead) larvae are now evident. f 168 h-old embryos. The distinction between eGFP⁺ (arrow) and eGFP^dim (arrowhead) larvae is even more substantial. g Fluorescence micrograph of an explant of colony 354–3. h Fluorescence micrograph of an explant of colony 354–5. Image input and output levels were adjusted to reveal eGFP fluorescence in stolonal mat. Inset: Image with levels scaled automatically in NIS-elements, showing intense fluorescence of developing oocytes (arrowhead). i Survivorship of eGFP⁺ and eGFP⁻ colonies from population 357. j Mat area measurements of colonies from population 357. All scale bars = 1 mm