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Series GSE262060 Query DataSets for GSE262060
Status Public on Mar 21, 2024
Title 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
Organisms Homo sapiens; synthetic construct
Experiment type Other
Summary 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.
Contributor(s) Shepherdson JL, Granas DM, Li J, Shariff Z, Plassmeyer SP, Holehouse AS, White MA, Cohen BA
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NIH grant(s)
Grant ID Grant title Affiliation Name
R01 GM092910 Analysis of combinatorial cis-regulation in synthetic and genomic promoters WASHINGTON UNIVERSITY Barak A Cohen
R21 HG012146 Cell-Based Assays For Deep Mutational Scans of Transcription Factors WASHINGTON UNIVERSITY Barak A Cohen
F30 EY033640 Multiplex functional assay of variant effect in the retinal transcription factor CRX WASHINGTON UNIVERSITY James Lewis Shepherdson
Submission date Mar 20, 2024
Last update date Mar 22, 2024
Contact name Barak Alon Cohen
Organization name Washington University School of Medicine
Department Genetics
Street address 4523 CLAYTON AVE CB #8510
State/province MO
ZIP/Postal code 63110
Country USA
Platforms (3)
GPL19424 Illumina NextSeq 500 (synthetic construct)
GPL34284 Illumina NovaSeq X Plus (Homo sapiens)
GPL34318 PacBio RS (synthetic construct)
Samples (18)
GSM8156809 Replicate 1, Fraction A
GSM8156810 Replicate 1, Fraction B
GSM8156811 Replicate 1, Fraction C
BioProject PRJNA1090111

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Supplementary file Size Download File type/resource
GSE262060_RAW.tar 388.6 Mb (http)(custom) TAR (of PARQUET, TSV)
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Raw data are available in SRA

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