Zebrafish Knockdown and Knockout

Injection of p53 morpholino (MO) or esco2 Cas9/guide RNA was performed on one-cell-stage zebrafish embryos at a concentration using 0.5 nl of 0.85 mM. Injected embryos were incubated at 28°C until the indicated stage and analyzed via brightfield microscopy. The sequence of p53 MO used to target exon 2 splice donor site of the p53 gene was 5′-CCCTTGCGAACTTACATCAAATTCT-3′. Cas9 mRNA was transcribed from the linearized pT3TS-nCas9n plasmid (Addgene) using the mMessage mMachine T3 kit (Life Technologies). Each RNA was purified using the RNeasy Kit (Qiagen). The CRISPR guide RNA was synthesized using the MegaShortScript T7 Kit (Life Technologies) and purified using the MegaClear Kit (Life Technologies). RNA concentration was quantified using the Nanodrop spectrophotometer. For CRISPR/Cas9 injections, 150 ng/µl of Cas9 mRNA and 30 ng/µl of RNA were used (Thomas et al., 2014 (link)).