The <scp>CRISPR</scp> ‐Cas9 knockout <i>DDC</i> <scp>SH</scp> ‐ <scp>SY</scp> 5 <scp>Y</scp> <i>in vitro</i> model for <scp>AADC</scp> deficiency provides insight into the pathogenicity of <scp>R</scp> 34 <scp>7Q</scp> and <scp>L</scp> 353 <scp>P</sc

Aromatic amino acid decarboxylase (AADC) deficiency is a severe inherited recessive neurotransmitter disorder caused by an impairment in dopamine synthesis due to the lack/modification of AADC, the enzyme converting l-dopa to dopamine. Patients exhibit severe movement disorders and neurodevelopmental delay, with a high risk of premature mortality. Given the lack of a reliable model for the disease, we developed a dopa decarboxylase knockout model using CRISPR/Cas9 technology in the SH-SY5Y neuroblastoma cell line. This model showed a deficiency in AADC protein and activity, with an altered dopamine metabolites profile (low homovanillic acid and high 3-O-methyldopa) and a modified expression of key enzymes, such as dopamine beta-hydroxylase and monoamine oxidases, which are involved in the catecholamine pathway. We then transfected the DDC-KO cells with two AADC catalytic variants, R347Q and L353P, which resulted in loss-of-function and an altered profile of dopamine metabolites. By combining several structural approaches (X-ray crystallography, molecular dynamics, small angle X-ray scattering, dynamic light scattering, and spectroscopy), we determined that both variants alter the flexibility of the structural element to which they belong, whose integrity is essential for catalysis. This change causes a mispositioning of essential residues at the active site, leading, in turn, to an unproductive external aldimine, identifying the molecular basis for the loss-of-function. Overall, the DDC-KO model recapitulates some key features of AADC deficiency, is useful to study the molecular basis of the disease, and represents an ideal system for small molecule screening regarding specific enzyme defects, paving the way for a precision therapeutic approach.