Functional activity scores of a DMS library representing coding single residue substitution variants in the transcription factor CRX measured in an engineered reporter cell line

Cone-Rod Homeobox, encoded by CRX, is a transcription factor (TF) essential for the terminal differentiation and maintenance of mammalian photoreceptors. 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. Overall design: A mutational scanning library was constructed containing all single residue substitution variants of human CRX, each marked by a unique sequence barcode (vBC) and a number of random barcodes (rBC). The library was integrated into a genomic landing pad in a HEK 293-derived cell line engineered to carry a CRX-responsive fluorescent transcriptional reporter. After selecting for successful integrants, cells were sorted into one of four bins based on fluorescence intensity (labeled A–D, lowest to highest fluorescence). Amplicon sequencing libraries were prepared from the sorted cells, and the frequence of each vBC was counted.