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| Status |
Public on Mar 13, 2021 |
| Title |
Massively parallel characterization of CYP2C9 variant enzyme activity and abundance |
| Organisms |
Escherichia coli; Saccharomyces cerevisiae; Homo sapiens |
| Experiment type |
Other
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| Summary |
CYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9 can affect patient response leading to an increased risk of hemorrhage. We developed Click-seq, a pooled yeast-based activity assay to test thousands of variants. Using Click-seq, we measured the activity of 6,142 missense variants expressed in yeast. We also measured the steady-state cellular abundance of 6,370 missense variants expressed in a human cell line using Variant Abundance by Massively Parallel sequencing (VAMP-seq). These data revealed that almost two-thirds of CYP2C9 variants showed decreased activity, and that protein abundance accounted for half of the variation in CYP2C9 function. We also measured activity scores for 319 previously unannotated human variants, many of which may have clinical relevance.
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| Overall design |
Barcoded single amino acid variant libraries of CYP2C9 variants were generated for yeast and human expression. A humanized yeast strain was transformed with the yeast codon-optimized CYP2C9 library, labeled with activity-based protein profiling, sorted for activity, and plasmid DNA extracted. Barcodes were amplified and counted with Illumina sequencing. The eGFP-tagged CYP2C9 library was recombined into HEK293T cells previously engineered to contain a Bxb1 recombination site at the AAVS1 locus. Upon sorting cells for APC or eGFP expression, genomic DNA was extracted. Barcodes were amplified and counted with Illumina sequencing. For both libraries, barcodes were associated back to the corresponding CYP2C9 variant by comparison with a barcode-variant map previously created by sequencing the variant library plasmids using PacBio sequencing.
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| Contributor(s) |
Amorosi CJ, Dunham MJ, Fowler DM |
| Citation(s) |
34314704 |
| |
| Submission date |
Jan 24, 2021 |
| Last update date |
Apr 28, 2023 |
| Contact name |
Clara Amorosi |
| Organization name |
University of Washington
|
| Department |
Genome Sciences
|
| Lab |
Dunham
|
| Street address |
3720 15th Ave. NE
|
| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platforms (5)
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| Samples (12)
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| Relations |
| BioProject |
PRJNA694476 |
| SRA |
SRP303073 |
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