Supplementary Figure 3: Development of dsxF^CRISPRh drive construct and its predicted homing process and molecular confirmation of the locus

(a) The drive construct (CRISPR^h cassette) contained the transcription unit of a human codon-optimised Cas9 controlled by the germline-restrictive zpg promoter, the RFP gene under the control of the neuronal 3xP3 promoter and the gRNA under the control of the constitutive U6 promoter, all enclosed within two attB sequences. The cassette was inserted at the target locus using recombinase-mediated cassette exchange (RMCE) by injecting embryos with a plasmid containing the cassette and a plasmid containing a ϕC31 recombination transcription unit. During meiosis the Cas9/gRNA complex cleaves the wild-type allele at the target locus (DSB) and the construct is copied across to the wild-type allele via HDR (homing) disrupting exon 5 in the process. (b) Representative example of molecular confirmation of successful RMCE events. Primers (blue arrows) that bind components of the CRISPR^h cassette were combined with primers that bind the genomic region surrounding the construct. PCRs were performed on both sides of the CRISPR^h cassette (5’ and 3’) on many individuals as well as wild-type controls (wt).