title: A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening.
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.
authors.affiliation: Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, California 92093, USA;
authors.forename: Yarui
authors.initials: Y
authors.lastname: Diao
authors.affiliation: Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, California 92093, USA;
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.affiliation: Department of Pharmacology and Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA;
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Meng
authors.affiliation: Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, California 92093, USA;
authors.forename: Inkyung
authors.initials: I
authors.lastname: Jung
authors.affiliation: Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, California 92093, USA;
authors.forename: Ah Young
authors.initials: AY
authors.lastname: Lee
authors.affiliation: Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, California 92093, USA; Medical Scientist Training Program, University of California, San Diego, La Jolla, California 92093, USA; Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, California 92093, USA;
authors.forename: Jesse
authors.initials: J
authors.lastname: Dixon
authors.affiliation: Institute for Human Genetics and Department of Neurology, University of California, San Francisco, San Francisco, California 94143, USA;
authors.forename: Lenka
authors.initials: L
authors.lastname: Maliskova
authors.affiliation: Department of Pharmacology and Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA;
authors.forename: Kun-Liang
authors.initials: KL
authors.lastname: Guan
authors.affiliation: Institute for Human Genetics and Department of Neurology, University of California, San Francisco, San Francisco, California 94143, USA;
authors.forename: Yin
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, California 92093, USA; Department of Cellular and Molecular Medicine, Institute of Genomic Medicine and Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Ren
mesh_terms: D064113:CRISPR-Cas Systems; D002460:Cell Line; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D018390:Gene Targeting / Q000379:methods; D005820:Genetic Testing / Q000379:methods; D015894:Genome, Human; D057166:High-Throughput Screening Assays; D006801:Humans; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D010641:Phenotype; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion
chemical_list: D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human
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