Fig. 2

CRISPR/Cas constructs and activity against white eye in the male germline. a Schematic representation of the transformation constructs. Cas9 or Cas12a coding sequences are under the transcriptional control of the male germline-specific β2-tubulin promoter (pβ2tub). The gRNA target sequences are under the control of the endogenous U6 Pol III promoter (pCcU6), and the constitutively expressed DsRed as a marker of transgenesis is under the control of the Ubiquitin promoter (pUb). Gypsy insulator sequences (gypsy) are indicated adjacent to pβ2tub and pUb. b Wild-type red-eye phenotype of an adult male medfly (left) and white-eye phenotype of a female medfly (right). c The percentage of white-eyed flies obtained by individually crossing hemizygous males expressing Cas9 and the gRNA targeting the white eye gene (Cas9.w) with white-eyed w2∆/w2∆ females (w) is shown in the top panel. As a control, hemizygous females (Cas9.w) were individually crossed with 10 white-eyed w2∆/w2∆ males (w). The middle panel shows the percentage of white-eyed flies obtained by individually crossing hemizygous males expressing Cas12a and the gRNA targeting the white eye gene (Cas12a.w) with white-eyed w2∆/w2∆ females (w), by crossing hemizygous males for Cas12a.m with white-eyed w2∆/w2∆ females (w) or by crossing hemizygous Cas12a.m females with w2∆/w2∆ white-eyed males (w). The bottom panel shows the control crosses between wild-type males with white-eyed females from the w2∆ strain (wt x w) and white-eyed males from the w2∆ strain crossed with wild-type females (w x wt). The numbers indicate the total number of individuals scored (N). d Diagram representation of the autosomal white eye gene of Ceratitis capitata, spanning six exons, and the sequence of the gRNA targeting exon 3 (PAM is shown in yellow). Below, the wild-type sequence is aligned to sequences obtained from F1 white-eyed flies, showing the indels induced by CRISPR/Cas9. The deletion spanning exon 2 of the w2∆ white eye mutant strain is indicated