Pneumatic picopump pv280

To increase the efficiency of the homology directed repair mechanism, the nonhomologous end joining repair mechanism was inhibited by a splice-blocking morpholino targeting ku70 (5′-AACTTTTTAGGCTCACCTGCATAGT-3′).¹⁹ (link)^,20 (link) A mix of 1 nL containing donor template (0.3 µM), gRNA (600 pg), Cas9 protein (5 ng), ku70 morpholino (1.5 ng), phenol-red (0.01%), and KCl 0.2 M was injected using a Pneumatic Picopump pv280 (World Precision Instruments) into zebrafish embryos at a one-cell stage. The injected embryos were grown into adulthood at 28°C in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄, supplemented with 0.01% methylene blue). Three months postfertilization, adult CRISPR-injected fish were in-crossed. The offspring was raised and individually genotyped. Subsequently, the appropriate znf408 mutants were crossed with Tg(flk1:GFP) fish to generate zebrafish with modified znf408 and a GFP reporter on the blood vessels (Fig. 1b). The generated fish were then in-crossed to generate homozygous models (Fig. 1b).

PMOs were designed by first assessing the target sequence for SRSF2 (SC35) ESE sites (threshold of 3.0) using the online ESE finder 3.0 tool.⁵² (link) Zebrafish ush2a exon 13-targeting PMOs were synthesized by Gene Tools (USA). PMOs were dissolved in ultrapure water at a stock concentration of 50 μg/μL and stored at −20°C. One nanoliter containing 0.5−4 pg per PMO and 0.25% (v/v) phenol red was injected into the yolk of 1- to 2-cell-stage embryos with a Pneumatic PicoPump pv280 (World Precision Instruments). After injection, embryos were raised at 28.5°C in E3 embryo medium until analysis. PMO sequences are provided in Table 1.