Mutational scanning of CRX classifies clinical variants and reveals biochemical properties of the transcriptional effector domain.

Cone-Rod Homeobox, encoded by CRX , is a transcription factor (TF) essential for the terminal differentiation and maintenance of mammalian photoreceptors. Structurally, CRX comprises an ordered DNA-binding homeodomain and an intrinsically disordered transcriptional effector domain. Although a handful of human variants in CRX have been shown to cause several different degenerative retinopathies with varying cone and rod predominance, as with most human disease genes the vast majority of observed CRX genetic variants are uncharacterized variants of uncertain significance (VUS). We performed a deep mutational scan (DMS) of nearly all possible single amino acid substitution variants in CRX, using an engineered cell-based transcriptional reporter assay. We measured the ability of each CRX missense variant to transactivate a synthetic fluorescent reporter construct in a pooled fluorescence-activated cell sorting assay and compared the activation strength of each variant to that of wild-type CRX to compute an activity score, identifying thousands of variants with altered transcriptional activity. We calculated a statistical confidence for each activity score derived from multiple independent measurements of each variant marked by unique sequence barcodes, curating a high-confidence list of nearly 2,000 variants with significantly altered transcriptional activity compared to wild-type CRX. We evaluated the performance of the DMS assay as a clinical variant classification tool using gold-standard classified human variants from ClinVar, and determined that activity scores could be used to identify pathogenic variants with high specificity. That this performance could be achieved using a synthetic reporter assay in a foreign cell type, even for a highly cell type-specific TF like CRX, suggests that this approach shows promise for DMS of other TFs that function in cell types that are not easily accessible. Per-position average activity scores closely aligned to a predicted structure of the ordered homeodomain and demonstrated position-specific residue requirements. The intrinsically disordered transcriptional effector domain, by contrast, displayed a qualitatively different pattern of substitution effects, following compositional constraints without specific residue position requirements in the peptide chain. The observed compositional constraints of the effector domain were consistent with the acidic exposure model of transcriptional activation. Together, the results of the CRX DMS identify molecular features of the CRX effector domain and demonstrate clinical utility for variant classification.