Extended Data Fig. 3: CRISPR–Cas9 knockout of Cirop in zebrafish and Xenopus laevis.
a, Real-time qPCR for cirop relative to actin in zebrafish embryos at indicated stages. cirop expression is only detectable using RNA extracted from tissue at the dorsal posterior side containing the DFCs. Data are mean ± s.e.m. ****P < 0.0001, Two-way ANOVA with Tukey test for multiple comparisons. 50%, 70% and 90% refer to epiboly stages. DFC, dorsal forerunner cells; hpf, hours postfertilization. n = 3 biological triplicates of 30-50 embryos. b, Depiction of genomic and protein structures of zebrafish Cirop. The sequence used is XM_002662823 using a START codon located 168 nucleotides upstream of the original one (light gray). Cirop protein domains are highlighted: the signal peptide (gray), the Zn+ catalytic domain (yellow), the cysteine-rich domain (red), and the transmembrane domain (blue). Yellow line, catalytic domain; green line, met-turn. The site and targeted sequence by the CRISPR gRNA is indicated in purple. Five different alleles were obtained with indicated mutations (green, insertion; red star, deletion), all leading to a frameshift (orange) with an early stop codon (underlined for mutation 1). While all mutations lead to the same LR phenotype in the homozygous state, line 1 was used for further investigation. c, Whole mount in situ hybridization for Cirop in 90% epiboly control and Cirop−/− zebrafish embryos. Scale bar, 0.1 mm. d, Depiction of genomic and protein structures of Xenopus laevis Cirop. A CRISPR gRNA was designed in the beginning of exon 4 (purple), leading to mutations with 92% efficiency. Chr., chromosome; SP, signal peptide; TM, transmembrane domain; wt, wild-type; Xl, Xenopus laevis.
