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LADL: Light-activated dynamic looping for endogenous gene expression control

Mayuri Rege, Ji Hun Kim, Jacqueline Valeri, Margaret C. Dunagin, Aryeh Metzger, Wanfeng Gong, Jonathan A. Beagan, Arjun Raj, View ORCID ProfileJennifer E. Phillips-Cremins
doi: https://doi.org/10.1101/349340
Mayuri Rege
1Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104
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Ji Hun Kim
1Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104
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Jacqueline Valeri
1Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104
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Margaret C. Dunagin
1Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104
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Aryeh Metzger
1Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104
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Wanfeng Gong
1Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104
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Jonathan A. Beagan
1Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104
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Arjun Raj
1Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104
2Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
3Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
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Jennifer E. Phillips-Cremins
1Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104
2Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
3Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
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  • ORCID record for Jennifer E. Phillips-Cremins
  • For correspondence: jcremins@seas.upenn.edu
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Abstract

Mammalian genomes are folded into tens of thousands of long-range looping interactions1,2. The cause and effect relationship between looping and genome function is poorly understood, and the extent to which chromatin loops are dynamic on short time scales remains a fundamental unanswered question. Currently available strategies for loop engineering involve synthetic transcription factors tethered to dCas93,4 or zinc fingers5,6, which are constitutively expressed5,6 or induced on long time scales by the presence of a small molecule3. Here we report a new class of 3-D optoepigenetic tools for the directed rearrangement of 3-D chromatin looping on short time scales using blue light. We create synthetic architectural proteins by fusing the CIBN protein subunit from Arabidopsis thaliana with enzymatically dead Cas9 (dCas9). We target our light-activated dynamic looping system (LADL) to two genomic anchors with CRISPR guide RNAs and engineer their spatial co-localization via light-induced heterodimerization of the cryptochrome 2 (CRY2) protein with dCas9-CIBN. We apply LADL to redirect a stretch enhancer (SE) away from its endogenous Klf4 target gene and to the Zfp462 promoter. Looping changes occur as early as four hours after light induction. Using single molecule RNA FISH, we observe a LADL-induced increase in the total nascent Zfp462 transcripts and the number of Zfp462 alleles expressing simultaneously per cell. Moreover, LADL also increased synchronous Sox2 expression after reinforcement of a known Sox2-SE looping interaction. LADL facilitates loop synchronization across a large population of cells without exogenous chemical cofactors and can enable future efforts to engineer reversible and oscillatory looping on short time scales.

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Posted June 18, 2018.
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LADL: Light-activated dynamic looping for endogenous gene expression control
Mayuri Rege, Ji Hun Kim, Jacqueline Valeri, Margaret C. Dunagin, Aryeh Metzger, Wanfeng Gong, Jonathan A. Beagan, Arjun Raj, Jennifer E. Phillips-Cremins
bioRxiv 349340; doi: https://doi.org/10.1101/349340
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LADL: Light-activated dynamic looping for endogenous gene expression control
Mayuri Rege, Ji Hun Kim, Jacqueline Valeri, Margaret C. Dunagin, Aryeh Metzger, Wanfeng Gong, Jonathan A. Beagan, Arjun Raj, Jennifer E. Phillips-Cremins
bioRxiv 349340; doi: https://doi.org/10.1101/349340

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