(A) Schematic of the de novo sphingolipid biosynthetic pathway and its feedback inhibition by ORMDLs through the sensing of ceramide levels. SPT, serine palmitoyltransferase; 3KDHSph, 3-keto-dihydrosphingosine; DHSph, dihydrosphingosine; DHCer, dihydroceramide; Cer, ceramide; Sph, sphingosine; S1P, sphingosine-1-phosphate. (B–D) Generation of Ormdl KO mice. Panels show the intron-exon organizations of the Ormdl genes and the protein coding regions (white). (B, C) Ormdl1 and Ormdl2 KO mice were produced by CRISPR/Cas9-induced mutations, resulting in frameshifts and premature stop codons. The locations of sgRNA sequences (red), PAM sites (green), as well as the changes in DNA and protein are indicated. The base insertion in the CRISPR/Cas9 modified Ormdl2 gene is underlined. (D) Ormdl3 KO mice were generated by germline Cre-LoxP recombination to excise exons 2, 3, and part of exon 4, resulting in the deletion of the entire protein-coding sequence. (E) RT-qPCR of Ormdl WT RNA in brain of Ormdl KO mice relative to that in WT mice. The mice were 8 weeks old. Probes detect the WT Ormdl sequences. Data are expressed as means ± SD. Unpaired Student’s t test; ***p<0.001. nd, not detectable. n = 4 for all genotypes. (F) Levels of dihydrosphingosine, total dihydroceramide, total ceramide, and sphingosine were determined by HPLC-tandem MS on lipid extracts of whole brains harvested from 8-week-old WT, Ormdl1 KO, Ormdl2 KO, Ormdl3 KO, Ormdl1/2 double KO, Ormdl1/3 double KO, and Ormdl2/3 double KO mice (Figure 1—source data 1). Data are expressed as means ± SD. One-way ANOVA with Bonferroni correction; *p<0.05, ***p<0.001. n = 8 for all genotypes. DKO, double knockout.