Transcriptional activity of an MPRA library containing sequences of genomic origin bound by the transcription factor CRX measured in retinas from mice carrying pathogenic CRX variants

Dozens of mutations in the photoreceptor-specific transcription factor CRX are linked with different human blinding diseases that vary in their severity and age of onset. How these mutations in a single TF cause a range of pathological phenotypes is not understood. We deployed massively parallel reporter assays (MPRAs) in live mouse retina to directly measure changes to CRX cis-regulatory function caused by two disease-causing mutations, one in the DNA binding domain (p.R90W) and the other in the activation domain (p.E168d2). Overall design: Two MPRA libraries were constructed containing 1,723 candidate cis-regulatory elment (CRE) sequences each 164 bp in length. The CREs were cloned upstream of one of two minimal promoters: rhodopsin (rho library) and hsp68 (hsp68 library). Each library was electroporated into mouse retinal explants from mice carrying each of six Crx genotypes: +/+, R90W/+, E168d2/+, R90W/R90W, E168d2/E168d2, -/-, with three biological replicates per combination. Additionally, each of the two input plasmid DNA libraries were sequenced.