Injection of Morpholino and sgRNA and Cas9
A splice-blocking morpholino for the boundary between intron 1 and exon 2 of zebrafish exosc9 was used (Genetools, 3′-actttatctgtgtaccgttttgtagCGCTTAGATGGGAGACAGACATACG-5′). Before injection, the morpholino and sgRNA was prepared. The sgRNA was diluted to 300 ng/μL with 2 μM Cas9 protein (NEB), 2M KCl, and 0.05% phenol red and heated to 37°C for 5 min. The morpholino was diluted in Danieau solution (0.4 mM MgSO4, 58 mM NaCl, 0.7 mM KCl, 5 mM HEPES, 0.6 mM Ca[NO3]2 [pH 7.6]) with phenol red and was heated to 65°C for 5 min.39 Freshly laid embryos were injected up until the two-cell stage with 1 nL of morpholino (20 ng) or 1 nL of guide RNA (gRNA). At least three clutches of embryos from different parents were used for each experiment.