Fig. 7.

Creation of the Eef1a^SF-P2A-tdTom allele. a Overview of Cas9-mediated insertional mutagenesis. Injection of Cas9:sgRNA_847 complexes and a plasmid containing a DNA repair template leads to the insertion of a SF-P2A-tdTomato cassette via homology directed repair. b Pedigree of tdTomato⁺ colonies in this paper. Colony 350–10 is a founder derived from an injected embryo and is a mosaic of edited tdTomato⁺ cells and wild-type tdTomato⁻ cells. c Left panel: light transmitted light micrograph of colony 350–10, showing gonozooids (white asterisk indicates the head). Arrows and arrowheads point to the same eggs as in the right panel. Right panel: Fluorescence micrograph of the same area of colony 350–10 showing tdTomato⁺ eggs. Single gonozooids contain oocytes that are fluorescent (arrow) and non-fluorescent (arrowheads). Scale bars = 1 mm. d Confocal image of one gonozooid, showing mixture of tdTomato positive and negative eggs. Scale bar = 100 μm. e PCR primers used to validate insertion at the Eef1a locus. Region matching the repair template is shown in gray box. f PCR amplification of upstream region from parental and tdTomato⁺ colonies. g Primer combinations tested in order to amplify the downstream region of Eef1a^SF-P2A-tdTom allele