Fig. 1.

Rationally chosen, native promoters drive strong expression of Cas9. Log₂ RPKM expression values for AAEL010097, AAEL007097, AAEL007584, AAEL005635, AAEL003877, and AAEL006511 were plotted across development. Samples include, from left to right: testis; male carcasses (lacking testes); carcasses of females before blood feeding (NBF), and at 12-, 24-, 36-, 48-, and 72-h postblood meal (PBM); ovaries from NBF females and at 12-, 24-, 36-, 48-, and 72-h PBM; embryos from 0–2 h through 72–76 h; whole larvae from first, second, third, and fourth instar; male pupae; female pupae (A). Genome browser snapshots of AAEL010097, AAEL007097, AAEL007584, AAEL005635, AAEL003877, and AAEL006511, including expression tracks for both 72-h PBM ovaries and testes. The light gray box indicates coding sequence; blue box represents promoter element with length indicated in bp; y axis shows the expression level based on raw read counts (B). Schematic representation of the piggybac-mediated Cas9 construct including the S. pyogenes Cas9-T2A-eGFP gene driven by selected promoters (blue), dsRed expressed under the control of the Opie2 promoter, which serves as a transgenesis marker. NLS represent nuclear localization signal (C). Confocal images using white light or GFP-filtering of transgenic Cas9 line ovaries (D). (Magnification, 200×.)