Generation of endogenously mNG-tagged centriolar proteins. (A i) Schematic illustration of the strategy to “knock-in” mNG at the N- or C-terminus of an endogenous locus; (L) is a short linker sequence. (ii) Images show the centriolar localization of the mNG-tagged CRISPR/Cas9-mediated knock-ins in living syncytial embryos (all images acquired in the early S-phase of nuclear cycle 12). N-terminally tagged mNG-Asl was not viable so it was expressed in a heterozygous (mNG-Asl/+) background. N-terminally tagged mNG-Plk4 consistently caused centriole overduplication (yellow arrows), so in subsequent experiments, we used a P-element insertion line of Plk4-mNG expressed from its endogenous promoter in the Plk4^−/− mutant background, (ePlk4-mNG, red dashed box). (B) Western blots show the expression levels of CRISPR/Cas9 knock-in lines and their cognate untagged endogenous proteins in 0–2 h old embryos. Prominent non-specific bands are highlighted (*); Actin, Cnn, and the Gaga transcription factor are shown as loading controls. A representative blot is shown from at least two technical repeats.