Transferman

To knockout the gene of interest, a 10–20 pL mixture of 100 ng/µL sgRNA and 200 ng/µL BE3 mRNA were microinjected into putative zygotes 10 h after IVF by using a micromanipulator (TransferMan, Eppendorf, Germany). In the control group, embryos were injected with the same amount of BE3 mRNA without sgRNA. To maximize the editing efficiency of the gene of interest, a cocktail of two or three sgRNAs was microinjected together with BE3 mRNA. Each sgRNA was kept at the same concentration (100 ng/µL).

Glass capillary needles were manufactured using glass capillaries (GC-100TF, Company) and vertical needle puller (model PB-7, Narishige). Solutions were injected into 4hpf zebrafish embryos using Femtojet microinjector (Eppendorf) and Transferman (Eppendorf) micromanipulator mounted to SteroLumar V12 stereomicroscope (Zeiss). The equipment was calibrated using injections of TMR_dextran (0.5 mg/ml in 90% DMSO, 2000 kDa TMR-dextran, Thermo Fischer) into halocarbon oil (Halocarbon oil 27, H8773, Sigma-Aldrich) and in into embryos followed by measurement of fluorescence intensities. Calibration by injections into halocarbon oil indicated delivery of 1.0 +/− 0.06 nl (n = 10), but fluorescence measurement from the embryos indicated that actual amount delivered in vivo was 2.0 +/− 1.2 nl (n = 10). In the toxicity experiment, either DMSO alone or MSNs dissolved in DMSO (10 mg/ml) were injected. After injections, damaged embryos were removed and healthy embryos were cultured in E3 at 28.5C.