High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis
Open accessAbstract
We present a method that harnesses massively parallel DNA synthesis and sequencing for the high-throughput functional analysis of regulatory sequences at single-nucleotide resolution. As a proof of concept, we quantitatively assayed the effects of all possible single-nucleotide mutations for three bacteriophage promoters and three mammalian core promoters in a single experiment per promoter. The method may also serve as a rapid screening tool for regulatory element engineering in synthetic biology.
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Massively parallel reporter assays and CRISPR genome editing have matured rapidly to enable functional characterization of targeted sequence perturbations at scale.
“To address this, there has been rapid maturation of technologies, such as massively parallel reporter assays121–123 and CRISPR genome editing, to support functional characterization of targeted sequence perturbations at scale.”
From A brief history of human disease genetics (2020) · cites this paper as [121]
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“For example, saturation mutagenesis (Melnikov et al., 2014; Patwardhan et al., 2009) can be used to screen all possible single-nucleotide variants in coding or non-coding regions, but relies on exogenous overexpression of the variant of interest, which may not always phenocopy endogenous variants,…”
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- Authors
- Patwardhan, R. P., Lee, C., Litvin, O., Young, D. L., Pe'er, D., & Shendure, J.
- Affiliations
- University of Washington (US)
- Columbia University (US)
- Corresponding author
- Patwardhan, Rupali P
- Shendure, Jay
- Venue
- Nature Biotechnology
- Published
- 2009-11-15
- Volume
- 27
- Issue
- 12
- Pages
- 1173-1175
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC2849652
- OpenAlex
- W2132224717
- Cited by
- 377
- References
- 16
- Citations by year
- 2026: 21 · 2025: 26 · 2024: 24 · 2023: 26 · 2022: 28 · 2021: 28 · 2020: 35 · 2019: 30 · 2018: 19 · 2017: 18 · 2016: 27 · 2015: 22 · 2014: 16 · 2013: 14 · 2012: 22
- FWCI
- 5.77
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Human Genome Research Institute (R21HG004749)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Keywords
- DNA regulatory elements, saturation mutagenesis, single-nucleotide resolution, promoter analysis, gene expression regulation, synthetic biology
- MeSH terms
- Algorithms, Base Sequence, DNA, Molecular Sequence Data, Mutagenesis, Site-Directed, Sequence Analysis, DNA, Regulatory Elements, Transcriptional
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