FIGURE 1.

Combination of the eyeless promoter and u ^S -Cas9 leads to the most efficient knock-out of norpA. (A) Scheme for the establishment of the tsCRISPR system in Drosophila eyes. (B) Immunoblot analysis assessing NorpA levels in various tissue specific norpA knock-outs. Genotypes of the driver constructs containing GMR, Ey or Rh1 promoters recombined with different Cas9 upstream variants are indicated above the panels. Single head extracts of 1–2 days old flies were analyzed and NorpA was detected by an α-NorpA antibody. α-Tubulin antibodies served as loading control. All lines were heterozygous for the eye specific promoter and Cas9. Molecular weight markers (kDa) are indicated on the left of each blot. (C) Quantification of the immunoblots shown in (B). Statistically significant differences in panel C (n = 3-5 for controls; n = 20 for knock-out flies) were analyzed by a one-way ANOVA test with Bonferroni correction (**** p < 0.0001, ns not significant). Error bars: SEM. NorpA signals were normalized to the tubulin signal and the NorpA level of the wild type was set to 100%. (D) Electroretinogram recordings to assess the knock-out efficiency of NorpA in the indicated genotype of 1–2 days old dark-adapted flies illuminated with one orange light pulse of 500 msec. (E) Quantification of the electroretinogram amplitudes shown in (D). Statistically significant differences in panel D (n = 15–21) were analyzed by a one-way ANOVA test with Bonferroni correction (**** p < 0.0001, ns not significant). Error bars: SEM.