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Orphan cytochrome P450 (CYP) enzymes are those for which biological substrates and function(s) are unknown. Cytochrome P450 20A1 (CYP20A1) is the last human orphan P450 enzyme, and orthologs occur as single genes in every vertebrate genome sequenced to date. The occurrence of high levels of  CYP20A1  transcripts in human substantia nigra and hippocampus and abundant maternal transcripts in zebrafish eggs strongly suggest roles both in the brain and during early embryonic development. Patients with chromosome 2 microdeletions including  CYP20A1  show hyperactivity and bouts of anxiety, among other conditions. Here, we created zebrafish  cyp20a1  mutants using CRISPR/Cas9, providing vertebrate models with which to study the role of CYP20A1 in behavior and other neurodevelopmental functions. The homozygous  cyp20a1  null mutants exhibited significant behavioral differences from wild-type zebrafish, both in larval and adult animals. Larval  cyp20a1 -/- mutants exhibited a strong increase in light-simulated movement (i.e., light–dark assay), which was interpreted as hyperactivity. Further, the larvae exhibited mild hypoactivity during the adaptation period of the optomotor assays. Adult  cyp20a1  null fish showed a pronounced delay in adapting to new environments, which is consistent with an anxiety paradigm. Taken together with our earlier morpholino  cyp20a1  knockdown results, the results described herein suggest that the orphan CYP20A1 has a neurophysiological role.

Orphan cytochrome P450 20a1 CRISPR/Cas9 mutants and neurobehavioral phenotypes in zebrafish