Generating X. tropicalis Transgenic Embryos

Wild-type X. tropicalis frogs were purchased from NASCO, and the snai2:eGFP transgenic line was generated previously^{23 (link)}. mRNAs encoding desired proteins were generated by in vitro transcription. For embryo production, female and male frogs were each primed with 20 IU human chorionic gonadotropin (HCG), and boosted with 200 IU HCG the next day for natural mating. Fertilized embryos were collected and injected in one blastomere at 2-cell stage; the uninjected blastomere served as a control. Alexa Fluor 555 (Invitrogen) or ß-galactosidase mRNA was co-injected as a lineage tracer. Morpholino for DPH1 KD (5’-CTTCCGCCATCTCTGACATATTTA-3’) was designed and synthesized by Gene Tools. The guide RNAs for CRISPR/Cas9 mediated DPH1 KO in X. tropicalis were designed using the CRISPRscan software^{52 (link)}. Three individual sites were designed for CRISPR targeting. Protospacers g1 (5′-GTGATGGGCGATGTGACGTA-3′), g2 (5′- GGTTGAAAGTTGAAGCGAA-3′) and g3 (5′- GGCATCAATCGGGACTGAG-3′) were embedded in forward primers. The DNA templates for gRNA transcription were obtained by PCR using pUC57-T7-gRNA scaffold vector as a template (see Supplementary Table 3 for primer sequences), and gRNAs were generated by in vitro transcription using the TranscriptAid T7 kit (Thermo Scientific K0441). To knock out dph1, each X. tropicalis embryo was injected with 1 ng Cas9 protein (PNA Bio) and 300 pg gRNA.