# Multiplex functional assay of variant effect in the retinal transcription factor CRX > **NIH NIH F30** · WASHINGTON UNIVERSITY · 2024 · $53,974 ## Abstract PROJECT SUMMARY The transcription factor Cone-Rod Homeobox (CRX ) is a master regulator of photoreceptor cell fate. Sequence variants in CRX can cause Retinitis Pigmentosa, Cone-Rod Dystrophy, and Leber Congenital Amaurosis, all inherited causes of vision loss and blindness. CRX is the only gene implicated in the pathogenesis of all three of these diseases, which present with both rod- and cone-centric phenotypes of varying age of onset and severity. Several CRX variants have been reported to cause severe dominant disease through antimorphic genetic interac- tions with wild-type CRX, and yet these mutations are adjacent to variants which are benign or only cause mild, recessive disease. Determining which mutations in CRX are pathogenic and quantifying their effect on functional activity is prerequisite to interpreting patient variation and predicting patient phenotypes. However, most variants in CRX are “Variants of Uncertain Significance” (VUS), meaning that insufficient clinical or functional evidence exists to determine their pathogenicity. Without a robust catalog of human genetic variation, advances in patient sequencing cannot be translated into clinical guidance or therapies for patients with uncharacterized variants. One potential solution to this challenge is Deep Mutational Scanning (DMS), which uses massive libraries of variant sequences in multiplexed assays to simultaneously measure the functional consequence of thousands of variants in a gene of interest in a single experiment. In DMS, each gene variant is assigned a quantitative functional pathogenicity score based on its activity in a molecular assay. This proposal will use DMS to simultaneously assay the transcriptional activity and abundance of all point substitutions, truncations, and frameshifts in CRX. The direct product of this work will be a “look-up table” listing the functional consequence of every CRX variant on protein activity and stability, which will be directly applicable to clinical variant classification and decision making. Given the retina's privileged location as a facile target for gene therapy, a catalog of human variation in CRX could inform the clinical management of patients afflicted with inherited retinopathies in the near term. Furthermore, this work will establish an extensible platform for additional DMS studies of other retinal transcription factors, broadly expanding our understanding of inherited retinal disease. ## Key facts - **NIH application ID:** 10789872 - **Project number:** 5F30EY033640-03 - **Recipient organization:** WASHINGTON UNIVERSITY - **Principal Investigator:** James Lewis Shepherdson - **Activity code:** F30 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2024 - **Award amount:** $53,974 - **Award type:** 5 - **Project period:** 2022-02-01 → 2026-01-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10789872 ## Citation > US National Institutes of Health, RePORTER application 10789872, Multiplex functional assay of variant effect in the retinal transcription factor CRX (5F30EY033640-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10789872. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*