Figure 2.

Figure 2.

Two tmem216 knockout zebrafish lines were generated by genome editing. Zebrafish knockout lines, tmem216^snyΔ175 and tmem216^snyR8Δ60, were generated by CRISPR/Cas9 genome editing. (A) Alignment of mutant genomic sequences with wildtype sequence from exon 3 to exon 4. The tmem216^snyΔ175 line features two deletions of two stretches of DNA of 172 and three nucleotides. The tmem216^snyR8Δ60 line features an eight-nucleotide repeat and deletion of two stretches of DNA of 56 and four nucleotides. Both mutations were expected to result in frameshift. (B) Protein sequence alignment between wildtype TMEM216, and the expected TMEM216^SNYΔ175 and TMEM216^SNYR8Δ60 mutant proteins. Note both mutations result in the loss of all four transmembrane domains. (C) RT-PCR of wild-type, heterozygous, and homozygous zebrafish, showing that homozygous knockouts express only mutant mRNA.