A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9 mediated genetic screening
- Yarui Diao1,
- Bin Li1,
- Zhipeng Meng2,
- Inkyung Jung1,
- Ahyoung Lee1,
- Jesse Dixon2,
- Lenka Maliskova3,
- Kun-Liang Guan2,
- Yin Shen3 and
- Bing Ren2,4
- 1 Ludwig Institute for Cancer Research;
- 2 University of California, San Diego;
- 3 University of California, San Francisco
- ↵* Corresponding author; email: biren{at}ucsd.edu
Abstract
With less than 2% of the human genome coding for proteins, a major challenge is to interpret the function of the non-coding DNA. Millions of regulatory sequences have been predicted in the human genome through the analyses 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 1Mbp POU5F1 locus in the 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 the 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 a high throughput screening for functional characterization of non-coding DNA, and reveal a previously unrecognized layer of gene regulation in human cells.
- Received July 18, 2015.
- Accepted January 20, 2016.
- Published by Cold Spring Harbor Laboratory Press
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