Generation of zebrafish TMEM55 KO lines

TMEM55B knockout (KO) mutants were generated using CRISPR/Cas9 methods^35,44. Briefly, specific gRNAs were co-injected with Cas9 mRNA in the yolk of TAB5 embryos at the 1-cell stage. At 24 h post fertilization (hpf) ~32 embryos from each injection were collected, the genomic DNA was extracted using the Extract-N-Amp Tissue PCR kit (Sigma-Aldrich, XNAT2) and the target efficacy was tested using Fluorescent-PCR methods⁴⁴. Only embryos from injections with high target frequency were moved into the system and grown until adulthood. The F₁ generations obtained from an outcross of potential F₀ founders with WT fish were screened for mutations and raised to adulthood. F₁ adults were genotyped by fin amputation, the gDNA was extracted by the fin tissues and fluorescent PCRs were performed³⁵. To determine the exact mutations in the F₁ population the fluorescent PCR reactions were Sanger sequenced and the exact sequence was reconstructed by manually annotation of the electropherograms using WT sequences as reference. Double-heterozygous TMEM55B-KO fish were in-crossed and double-homozygous animals were selected by genotyping. Finally, TMEM55B-KO were incrossed and the embryos were injected with specific gRNAs targeting the TMEM55A gene to obtain the triple-KO animals. For a list of the targets used see Supplementary Table ¹.