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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.

SUBMITTER: Diao Y 

PROVIDER: S-EPMC4772021 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening.

Diao Yarui Y   Li Bin B   Meng Zhipeng Z   Jung Inkyung I   Lee Ah Young AY   Dixon Jesse J   Maliskova Lenka L   Guan Kun-Liang KL   Shen Yin Y   Ren Bing B  

Genome research 20160126 3


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 1  ...[more]

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