Fig 2. Flowchart showing generation and identification of ECFP knockout mutants based on transgenic PUbB2 P61 x HWE hybrids.

Five groups of Cas9/sgRNA constructs were each micro-injected into ~500–700 double-hemizygous embryos (G₀). All surviving G₀ individuals were outcrossed to HWE and pooled. All G₁ individuals of each pool were screened for the ECFP knockout phenotype. Pools containing individuals with ECFP knockout phenotype were again outcrossed to HWE to generate G₂. DNA was extracted and sequenced from G₀ adults and from G₁ and G₂ larvae showing ECFP knockout phenotype and also from groups of G₁ larvae, which did not show a mutant phenotype.