High-throughput mapping of regulatory DNA
Open accessAbstract
Quantifying the effects of cis-regulatory DNA on gene expression is a major challenge. Here, we present the multiplexed editing regulatory assay (MERA), a high-throughput CRISPR-Cas9-based approach that analyzes the functional impact of the regulatory genome in its native context. MERA tiles thousands of mutations across ∼40 kb of cis-regulatory genomic space and uses knock-in green fluorescent protein (GFP) reporters to read out gene activity. Using this approach, we obtain quantitative information on the contribution of cis-regulatory regions to gene expression. We identify proximal and distal regulatory elements necessary for expression of four embryonic stem cell-specific genes. We show a consistent contribution of neighboring gene promoters to gene expression and identify unmarked regulatory elements (UREs) that control gene expression but do not have typical enhancer epigenetic or chromatin features. We compare thousands of functional and nonfunctional genotypes at a genomic location and identify the base pair-resolution functional motifs of regulatory elements.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
A shared limitation of single-gene screens is that phenotyping is restricted to one or a few genes per experiment.
“A shared limitation of single-gene screens, whether by CRISPR, CRISPRi, or CRISPRa, is that the phenotyping is restricted to one or a few genes per experiment, e.g. by engineering a reporter to the target gene133,201,203,208, by labelling mRNA products with fluorescence in situ hybridization (FISH)209, or by focusing on drug-responsive202,204, antibody-detectable92, or proliferation-related105,200”
From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [201]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116 · see citing works
Multiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339 · see citing works
Needleman, S. B., & Wunsch, C. D. (1970) · Journal of Molecular Biology · doi:10.1016/0022-2836(70)90057-4 · Cited by 11,288 · see citing works
Topological domains in mammalian genomes identified by analysis of chromatin interactions
Dixon, J. R., Selvaraj, S., Yue, F., Kim, A., Li, Y., Shen, Y., Hu, M., Liu, J. S., & Ren, B. (2012) · Nature · doi:10.1038/nature11082 · Cited by 6,829 · see citing works
Genome-scale CRISPR-Cas9 knockout screening in human cells
Shalem, O., Sanjana, N. E., Hartenian, E., Shi, X., Scott, D. A., Mikkelsen, T. S., Heckl, D., Ebert, B. L., Root, D. E., Doench, J. G., et al. (2014) · Science · doi:10.1126/science.1247005 · Cited by 5,234 · see citing works
CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes
Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z., Brar, G. A., Torres, S. E., Stern-Ginossar, N., Brandman, O., Whitehead, E. H., Doudna, J. A., et al. (2013) · Cell · doi:10.1016/j.cell.2013.06.044 · Cited by 3,696 · see citing works
High-Frequency off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells
Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander, J. D. (2013) · Nature Biotechnology · doi:10.1038/nbt.2623 · Cited by 3,292 · see citing works
Histone modifications at human enhancers reflect global cell-type-specific gene expression
Heintzman, N. D., Hon, G. C., Hawkins, R. D., Kheradpour, P., Stark, A., Harp, L. F., Ye, Z., Lee, L. K., Stuart, R. K., Ching, C. W., et al. (2009) · Nature · doi:10.1038/nature07829 · Cited by 2,533 · see citing works
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases
Tsai, S. Q., Zheng, Z., Nguyen, N. T., Liebers, M., Topkar, V. V., Thapar, V., Wyvekens, N., Khayter, C., Iafrate, A. J., Le, L. P., et al. (2015) · Nature Biotechnology · doi:10.1038/nbt.3117 · Cited by 2,268 · see citing works
A unique chromatin signature uncovers early developmental enhancers in humans
Rada-Iglesias, A., Bajpai, R., Swigut, T., Brugmann, S. A., Flynn, R. A., & Wysocka, J. (2011) · Nature · doi:10.1038/nature09692 · Cited by 2,233 · see citing works
Improving CRISPR-Cas nuclease specificity using truncated guide RNAs
Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2808 · Cited by 1,993 · see citing works
Engineered CRISPR-Cas9 nucleases with altered PAM specificities
Kleinstiver, B. P., Prew, M. S., Tsai, S. Q., Topkar, V. V., Nguyen, N. T., Zheng, Z., Gonzales, A. P. W., Li, Z., Peterson, R. T., Yeh, J.-R. J., et al. (2015) · Nature · doi:10.1038/nature14592 · Cited by 1,624 · see citing works
Genome-Wide Quantitative Enhancer Activity Maps Identified by STARR-seq
Arnold, C. D., Gerlach, D., Stelzer, C., Boryń, Ł. M., Rath, M., & Stark, A. (2013) · Science · doi:10.1126/science.1232542 · Cited by 1,125 · see citing works
Koike-Yusa, H., Li, Y., Tan, E.-P., Velasco-Herrera, M. D. C., & Yusa, K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2800 · Cited by 1,064 · see citing works
Functional footprinting of regulatory DNA
Vierstra, J., Reik, A., Chang, K.-H., Stehling-Sun, S., Zhou, Y., Hinkley, S. J., Paschon, D. E., Zhang, L., Psatha, N., Bendana, Y. R., et al. (2015) · Nature Methods · doi:10.1038/nmeth.3554 · Cited by 133 · see citing works
Flagged first, then by citations
Research works printed but not linked: 29
- [1]Jenuwein T Allis CD Translating the histone code Science 2001 293 1074 1080 11498575
- [2]Bernstein BE A bivalent chromatin structure marks key developmental genes in embryonic stem cells Cell 2006 125 315 326 16630819
- [5]Creyghton MP Histone H3K27ac separates active from poised enhancers and predicts developmental state Proceedings of the National Academy of Sciences of the United States of America 2010 107 21931 21936 21106759 PMC3003124
- [6]Melnikov A Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay Nature biotechnology 2012 30 271 277 PMC3297981 22371084
- [8]Patwardhan RP Massively parallel functional dissection of mammalian enhancers in vivo Nature biotechnology 2012 30 265 270 PMC3402344 22371081
- [9]Fullwood MJ An oestrogen-receptor-alpha-bound human chromatin interactome Nature 2009 462 58 64 19890323 PMC2774924
- [10]Lieberman-Aiden E Comprehensive mapping of long-range interactions reveals folding principles of the human genome Science 2009 326 289 293 19815776 PMC2858594
- [11]Simonis M Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C) Nature genetics 2006 38 1348 1354 17033623
- [12]Dostie J Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements Genome research 2006 16 1299 1309 16954542 PMC1581439
- [13]Kwasnieski JC Fiore C Chaudhari HG Cohen BA High-throughput functional testing of ENCODE segmentation predictions Genome research 2014 24 1595 1602 25035418 PMC4199366
- [16]Mali P RNA-guided human genome engineering via Cas9 Science 2013 339 823 826 23287722 PMC3712628
- [17]Jinek M RNA-programmed genome editing in human cells eLife 2013 2 e00471 23386978 PMC3557905
- [19]Cho SW Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases Genome research 2014 24 132 141 24253446 PMC3875854
- [23]Cradick TJ Fine EJ Antico CJ Bao G CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity Nucleic acids research 2013 41 9584 9592 23939622 PMC3814385
- [24]Wang T Wei JJ Sabatini DM Lander ES Genetic screens in human cells using the CRISPR-Cas9 system Science 2014 343 80 84 24336569 PMC3972032
- [26]Zhou Y High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells Nature 2014 509 487 491 24717434
- [28]Chen S Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis Cell 2015 160 1246 1260 25748654 PMC4380877
- [29]Arbab M Srinivasan S Hashimoto T Geijsen N Sherwood Richard I Cloning-free CRISPR Stem Cell Reports PMC4649464 26527385
- [30]Young RA Control of the embryonic stem cell state Cell 2011 144 940 954 21414485 PMC3099475
- [31]Yue F A comparative encyclopedia of DNA elements in the mouse genome Nature 2014 515 355 364 25409824 PMC4266106
- [32]Singh AM Hamazaki T Hankowski KE Terada N A heterogeneous expression pattern for Nanog in embryonic stem cells Stem cells 2007 25 2534 2542 17615266
- [33]Rajagopal N RFECS: a random-forest based algorithm for enhancer identification from chromatin state PLoS Comput Biol 2013 9 e1002968 23526891 PMC3597546
- [34]Consortium EP An integrated encyclopedia of DNA elements in the human genome Nature 2012 489 57 74 22955616 PMC3439153
- [35]John S Chromatin accessibility pre-determines glucocorticoid receptor binding patterns Nature genetics 2011 43 264 268 21258342 PMC6386452
- [36]Sherwood RI Discovery of directional and nondirectional pioneer transcription factors by modeling DNase profile magnitude and shape Nature biotechnology 2014 32 171 178 PMC3951735 24441470
- [37]Guo Y Mahony S Gifford DK High resolution genome wide binding event finding and motif discovery reveals transcription factor spatial binding constraints PLoS computational biology 2012 8 e1002638 22912568 PMC3415389
- [40]Leung D Integrative analysis of haplotype-resolved epigenomes across human tissues Nature 2015 518 350 354 25693566 PMC4449149
- [41]Li G Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation Cell 2012 148 84 98 22265404 PMC3339270
- [42]Woo YH Walker M Churchill GA Coordinated expression domains in mammalian genomes PloS one 2010 5 e12158 20805879 PMC2923606
Cited by
Sorted by citations
Activity-by-contact model of enhancer–promoter regulation from thousands of CRISPR perturbations
Fulco, C. P., Nasser, J., Jones, T. R., Munson, G., Bergman, D. T., Subramanian, V., Grossman, S. R., Anyoha, R., Doughty, B. R., Patwardhan, T. A., et al. (2019) · Nature Genetics · doi:10.1038/s41588-019-0538-0 · Cited by 1,111 · see citing works
Long-range enhancer–promoter contacts in gene expression control
Schoenfelder, S., & Fraser, P. (2019) · Nature Reviews Genetics · doi:10.1038/s41576-019-0128-0 · Cited by 1,102 · see citing works
Javierre, B. M., Burren, O. S., Wilder, S. P., Kreuzhuber, R., Hill, S. M., Sewitz, S., Cairns, J., Wingett, S. W., Várnai, C., Thiecke, M. J., et al. (2016) · Cell · doi:10.1016/j.cell.2016.09.037 · Cited by 1,046 · see citing works
Systematic mapping of functional enhancer–promoter connections with CRISPR interference
Fulco, C. P., Munschauer, M., Anyoha, R., Munson, G., Grossman, S. R., Perez, E. M., Kane, M., Cleary, B., Lander, E. S., & Engreitz, J. M. (2016) · Science · doi:10.1126/science.aag2445 · Cited by 643 · see citing works
A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens
Gasperini, M., Hill, A. J., McFaline-Figueroa, J. L., Martin, B., Kim, S., Zhang, M. D., Jackson, D., Leith, A., Schreiber, J., Noble, W. S., et al. (2019) · Cell · doi:10.1016/j.cell.2018.11.029 · Cited by 613 · see citing works
Klann, T. S., Black, J. B., Chellappan, M., Safi, A., Song, L., Hilton, I. B., Crawford, G. E., Reddy, T. E., & Gersbach, C. A. (2017) · Nature Biotechnology · doi:10.1038/nbt.3853 · Cited by 447 · see citing works
Towards a comprehensive catalogue of validated and target-linked human enhancers
Gasperini, M., Tome, J. M., & Shendure, J. (2020) · Nature Reviews Genetics · doi:10.1038/s41576-019-0209-0 · Cited by 396 · see citing works
Multiplexed Engineering and Analysis of Combinatorial Enhancer Activity in Single Cells
Xie, S., Duan, J., Li, B., Zhou, P., & Hon, G. C. (2017) · Molecular Cell · doi:10.1016/j.molcel.2017.03.007 · Cited by 334 · see citing works
A compendium of promoter-centered long-range chromatin interactions in the human genome
Jung, I., Schmitt, A., Diao, Y., Lee, A. J., Liu, T., Yang, D., Tan, C., Eom, J., Chan, M., Chee, S., et al. (2019) · Nature Genetics · doi:10.1038/s41588-019-0494-8 · Cited by 362 · see citing works
CRISPR/Cas9-based engineering of the epigenome
Pulecio, J., Verma, N., Mejía-Ramírez, E., Huangfu, D., & Raya, A. (2017) · Cell Stem Cell · doi:10.1016/j.stem.2017.09.006 · Cited by 300 · see citing works
Discovery of stimulation-responsive immune enhancers with CRISPR activation
Simeonov, D. R., Gowen, B. G., Boontanrart, M., Roth, T. L., Gagnon, J. D., Mumbach, M. R., Satpathy, A. T., Lee, Y., Bray, N. L., Chan, A. Y., et al. (2017) · Nature · doi:10.1038/nature23875 · Cited by 297 · see citing works
Determinants of Base Editing Outcomes from Target Library Analysis and Machine Learning
Arbab, M., Shen, M. W., Mok, B., Wilson, C., Matuszek, Ż., Cassa, C. A., & Liu, D. R. (2020) · Cell · doi:10.1016/j.cell.2020.05.037 · Cited by 294 · see citing works
Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes
Hess, G. T., Tycko, J., Yao, D., & Bassik, M. C. (2017) · Molecular Cell · doi:10.1016/j.molcel.2017.09.029 · Cited by 234 · see citing works
Genome-scale high-resolution mapping of activating and repressive nucleotides in regulatory regions
Ernst, J., Melnikov, A., Zhang, X., Wang, L., Rogov, P., Mikkelsen, T. S., & Kellis, M. (2016) · Nature Biotechnology · doi:10.1038/nbt.3678 · Cited by 162 · see citing works
Gasperini, M., Findlay, G. M., McKenna, A., Milbank, J. H., Lee, C., Zhang, M. D., Cusanovich, D. A., & Shendure, J. (2017) · The American Journal of Human Genetics · doi:10.1016/j.ajhg.2017.06.010 · Cited by 143 · see citing works
Global reorganisation of cis-regulatory units upon lineage commitment of human embryonic stem cells
Freire-Pritchett, P., Schoenfelder, S., Várnai, C., Wingett, S. W., Cairns, J., Collier, A. J., García-Vílchez, R., Furlan-Magaril, M., Osborne, C. S., Fraser, P., et al. (2017) · eLife · doi:10.7554/elife.21926 · Cited by 161 · see citing works
“However, a regulatory function for some ‘unmarked’ PIRs also cannot be ruled out as recent mutagenesis experiments have identified functional elements that lack ’classic’ chromatin annotations (Pradeepa et al., 2016; Rajagopal et al., 2016).”
BackgroundZacher, B., Michel, M., Schwalb, B., Cramer, P., Tresch, A., & Gagneur, J. (2017) · PLOS ONE · doi:10.1371/journal.pone.0169249 · Cited by 91 · see citing works
- Authors
- Rajagopal, N., Srinivasan, S., Kooshesh, K., Guo, Y., Edwards, M. D., Banerjee, B., Syed, T., Emons, B. J. M., Gifford, D. K., & Sherwood, R. I.
- Affiliations
- MIT Computer Science and Artificial Intelligence Laboratory (US)
- Brigham and Women's Hospital (US)
- Corresponding author
- Gifford, David K
- Sherwood, Richard I
- Venue
- Nature Biotechnology
- Published
- 2016-01-25
- Volume
- 34
- Issue
- 2
- Pages
- 167-174
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC5108523
- OpenAlex
- W2254864826
- Cited by
- 238
- References
- 15
- Citations by year
- 2026: 9 · 2025: 9 · 2024: 11 · 2023: 17 · 2022: 15 · 2021: 25 · 2020: 23 · 2019: 30 · 2018: 35 · 2017: 42 · 2016: 22
- FWCI
- 22.36
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
- PMC
- Funders
- National Human Genome Research Institute (U01HG007037, R01HG008754, R01HG008363)
- National Institute of Diabetes and Digestive and Kidney Diseases (K01DK101684)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
- Keywords
- gene expression regulation, CRISPR/Cas9 mutagenesis, enhancers, promoter activity, GFP reporter, functional motifs, high-throughput functional genomics
- MeSH terms
- Animals, Chromosome Mapping, Gene Expression Regulation, Sequence Analysis, DNA, Genomics, Regulatory Elements, Transcriptional, Mice, CRISPR-Cas Systems
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
