A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening
- Yarui Diao1,7,
- Bin Li1,
- Zhipeng Meng2,
- Inkyung Jung1,
- Ah Young Lee1,
- Jesse Dixon1,3,4,
- Lenka Maliskova5,
- Kun-liang Guan2,
- Yin Shen5,7 and
- Bing Ren1,6
- 1Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, California 92093, USA;
- 2Department of Pharmacology and Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA;
- 3Medical Scientist Training Program, University of California, San Diego, La Jolla, California 92093, USA;
- 4Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, California 92093, USA;
- 5Institute for Human Genetics and Department of Neurology, University of California, San Francisco, San Francisco, California 94143, USA;
- 6Department of Cellular and Molecular Medicine, Institute of Genomic Medicine and Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA
- Corresponding authors: yin.shen{at}ucsf.edu, biren{at}ucsd.edu
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↵7 These authors contributed equally to this work.
Abstract
With <2% of the human genome coding for proteins, a major challenge is to interpret the function of the noncoding DNA. Millions of regulatory sequences have been predicted in the human genome through analysis of DNA methylation, chromatin modification, hypersensitivity to nucleases, and transcription factor binding, but few have been shown to regulate transcription in their native contexts. We have developed a high-throughput CRISPR/Cas9-based genome-editing strategy and used it to interrogate 174 candidate regulatory sequences within the 1-Mbp POU5F1 locus in human embryonic stem cells (hESCs). We identified two classical regulatory elements, including a promoter and a proximal enhancer, that are essential for POU5F1 transcription in hESCs. Unexpectedly, we also discovered a new class of enhancers that contribute to POU5F1 transcription in an unusual way: Disruption of such sequences led to a temporary loss of POU5F1 transcription that is fully restored after a few rounds of cell division. These results demonstrate the utility of high-throughput screening for functional characterization of noncoding DNA and reveal a previously unrecognized layer of gene regulation in human cells.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.197152.115.
- Received July 18, 2015.
- Accepted January 20, 2016.
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
