Article | Published:

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

Abstract

Mammalian genomes are folded into tens of thousands of long-range looping interactions. The cause-and-effect relationship between looping and genome function is poorly understood, and the extent to which loops are dynamic on short time scales remains an unanswered question. Here, we engineer a new class of synthetic architectural proteins for directed rearrangement of the three-dimensional genome using blue light. We target our light-activated-dynamic-looping (LADL) system to two genomic anchors with CRISPR guide RNAs and induce their spatial colocalization via light-induced heterodimerization of cryptochrome 2 and a dCas9-CIBN fusion protein. We apply LADL to redirect a stretch enhancer (SE) away from its endogenous Klf4 target gene and to the Zfp462 promoter. Using single-molecule RNA–FISH, we demonstrate that de novo formation of the Zfp462-SE loop correlates with a modest increase in Zfp462 expression. LADL facilitates colocalization of genomic loci without exogenous chemical cofactors and will enable future efforts to engineer reversible and oscillatory loops on short time scales.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

The 5C data from this study have been submitted to the NCBI Gene Expression Omnibus under accession number GSE115963. Custom code for full reproducibility of all analyses is available upon request.

References

  1. 1.

    Phanstiel, D. H. et al. Static and dynamic DNA loops form AP-1-bound activation hubs during macrophage development. Mol. Cell 67, 1037–1048 e1036 (2017).

  2. 2.

    Rao, S. S. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665–1680 (2014).

  3. 3.

    Morgan, S. L. et al. Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping. Nat. Commun. 8, 15993 (2017).

  4. 4.

    Hao, N., Shearwin, K. E. & Dodd, I. B. Programmable DNA looping using engineered bivalent dCas9 complexes. Nat. Commun. 8, 1628 (2017).

  5. 5.

    Deng, W. et al. Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor. Cell 149, 1233–1244 (2012).

  6. 6.

    Deng, W. et al. Reactivation of developmentally silenced globin genes by forced chromatin looping. Cell 158, 849–860 (2014).

  7. 7.

    Liu, H. et al. Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis. Science 322, 1535–1539 (2008).

  8. 8.

    Bugaj, L. J., Choksi, A. T., Mesuda, C. K., Kane, R. S. & Schaffer, D. V. Optogenetic protein clustering and signaling activation in mammalian cells. Nat. Methods 10, 249 (2013).

  9. 9.

    Kennedy, M. J. et al. Rapid blue-light-mediated induction of protein interactions in living cells. Nat. Methods 7, 973–975 (2010).

  10. 10.

    Konermann, S. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).

  11. 11.

    Ozkan-Dagliyan, I. et al. Formation of Arabidopsis cryptochrome 2 photobodies in mammalian nuclei: application as an optogenetic DNA damage checkpoint switch. J. Biol. Chem. 288, 23244–23251 (2013).

  12. 12.

    Che, D. L., Duan, L., Zhang, K. & Cui, B. The dual characteristics of light-induced cryptochrome 2, homo-oligomerization and heterodimerization, for optogenetic manipulation in mammalian cells. ACS Synth. Biol. 4, 1124–1135 (2015).

  13. 13.

    Polstein, L. R. & Gersbach, C. A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198–200 (2015).

  14. 14.

    Beagan, J. A. et al. Local genome topology can exhibit an incompletely rewired 3D-folding state during somatic cell reprogramming. Cell Stem Cell 18, 611–624 (2016).

  15. 15.

    Beagan, J. A. et al. YY1 and CTCF orchestrate a 3D chromatin looping switch during early neural lineage commitment. Genome Res. 27, 1139–1152 (2017).

  16. 16.

    Dostie, J. et al. Chromosome conformation capture carbon copy (5C): a massively parallel solution for mapping interactions between genomic elements. Genome Res. 16, 1299–1309 (2006).

  17. 17.

    Phillips-Cremins, J. E. et al. Architectural protein subclasses shape 3D organization of genomes during lineage commitment. Cell 153, 1281–1295 (2013).

  18. 18.

    Kim, J. H. et al. 5C-ID: increased resolution chromosome-conformation-capture-carbon-copy with in situ 3C and double alternating primer design. Methods 142, 39–46 (2018).

  19. 19.

    Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).

  20. 20.

    Sakuma, T., Nishikawa, A., Kume, S., Chayama, K. & Yamamoto, T. Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sci. Rep. 4, 5400 (2014).

  21. 21.

    Cheng, A. W. et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23, 1163 (2013).

  22. 22.

    Taslimi, A. et al. An optimized optogenetic clustering tool for probing protein interaction and function. Nat. Commun. 5, 4925 (2014).

  23. 23.

    Hsu, S. C. et al. The BET protein BRD2 cooperates with CTCF to enforce transcriptional and architectural boundaries. Mol. Cell 66, 102–116 (2017).

  24. 24.

    Lajoie, B. R., van Berkum, N. L., Sanyal, A. & Dekker, J. My5C: web tools for chromosome conformation capture studies. Nat. Methods 6, 690–691 (2009).

  25. 25.

    Hnisz, D. et al. Activation of proto-oncogenes by disruption of chromosome neighborhoods. Science 351, 1454–1458 (2016).

  26. 26.

    Imakaev, M. et al. Iterative correction of Hi-C data reveals hallmarks of chromosome organization. Nat. Methods 9, 999–1003 (2012).

  27. 27.

    Gilgenast, T. G. & Phillips-Cremins, J. E. Systematic evaluation of statistical methods for identifying looping interactions in 5C data. Cell Syst. 8, 197–211 (2019).

  28. 28.

    Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

Download references

Acknowledgements

We thank members of the Cremins lab for helpful discussions. J.E.P.C. is a New York Stem Cell Foundation–Robertson Investigator and an Alfred P. Sloan Foundation Fellow. This research was supported by The New York Stem Cell Foundation (J.E.P.C), the Alfred P. Sloan Foundation (J.E.P.C), the NIH Director’s New Innovator Award from the National Institute of Mental Health (grant no. 1DP2MH11024701 to J.E.P.C), a 4D Nucleome Common Fund grant (no. 1U01HL12999801 to J.E.P.C), a joint NSF-NIGMS grant to support research at the interface of the biological and mathematical sciences (no. 1562665 to J.E.P.C) and a National Science Foundation Graduate Research Fellowship (grant no. DGE-1321851 to J.A.B.).

Author information

J.E.P.C., M.R., J.V. and A.M. conceptualized the system. M.R., J.H.K., J.V. and W.G. designed and performed the experiments. M.C.D. and A.R. designed and conducted RNA–FISH experiments. M.C.D., A.R. and J.E.P.C. analyzed FISH data. J.H.K., J.A.B., K.R.T., T.G.G. and J.E.P.C. performed the 5C data analysis. J.E.P.C. wrote the manuscript with help from all other authors.

Correspondence to Jennifer E. Phillips-Cremins.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information: Nicole Rusk was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Overview of LADL components.

Functional light-induced looping requires three components: the dCas9-CIBN Anchor protein, the CRY2 Bridge and the gRNAs as the Target. We combined these components in a two-plasmid system as follows: (a) the LADL Anchor plasmid with the dCas9-CIBN Anchor protein co-transfected with the LADL Bridge + Target plasmid that has both the CRY2 Bridge and the gRNAs as Target. This plasmid combination is referred to as “LADL (Anchor + Bridge + Target)”. The three negative control combinations exclude one critical component each: (b) The “Empty anchor control” excludes the dCas9-CIBN Anchor protein but includes the CRY2 Bridge and the gRNAs as Target. (c) The “Empty target control” excludes the gRNA expression module but includes the dCas9-CIBN Anchor and CRY2 Bridge. (d) The “Empty bridge control” excludes the soluble CRY2 Bridge but includes the dCas9-CIBN Anchor and the gRNAs as Target. (e) The “One-sided gRNA control” excludes the two gRNAs targeting the enhancer but includes the two gRNAs targeting the promoter, the soluble CRY2 Bridge and the dCas9-CIBN Anchor. For comprehensive plasmid maps please refer to Supplementary Figure 2.

Supplementary Figure 2 Complete list of intermediate and final LADL plasmids.

(a) Plasmid S13.1. Backbone plasmid used to create the Anchor plasmids. Ampicillin resistant. (b) LADL Anchor plasmid with the dCas9-CIBN. CRY2/CIBN heterodimerization based Anchor plasmid with puromycin resistance. The relevant parts are: EF1A promoter (EF1α); three copies of the FLAG tag (3XFLAG); α – importin Nuclear Localization Signal (NLS); Cryptochrome 2 photolyase homology region (CRY2PHR); SV40 Nuclear Localization Signal (NLS); Glycine-Serine Linker (GS); SV40 Nuclear Localization Signal (NLS); dCas9 D10A H840A; Glycine-Serine Linker (GS); α – importin Nuclear Localization Signal (NLS); Cryptochrome 2 photolyase homology region (CRY2PHR); SV40 Nuclear Localization Signal (NLS); 2A self-cleaving peptide (2A); puromycin resistance (Puro). This plasmid was used in Figures 15 and Supplementary Figures 1, 411, 13 and is referred to as the “LADL Anchor” plasmid. (c) Empty anchor control plasmid. Puromycin resistance gene expressed from the EF1a promoter without any dCas9-CIBN Anchor protein. This plasmid was used as the puromycin resistant plasmid in the ‘Empty anchor control’ in Figures 12 and Supplementary Figures 1, 5, 710. (d) Plasmid S12.1. The modified Yamamoto Ampicillin resistant plasmid (Addgene #58768) capable of cloning a single gRNA when cut with BbsI and multiplexing four gRNAs after Golden Gate assembly with the companion B1, B2, B3 Spectinomycin resistant plasmids. This plasmid was used as a backbone to clone gRNA 129 shown in (e). (e) Single gRNA 129 cloned into the Ampicillin resistant S12.1 plasmid backbone using BbsI digestion. (f-h) Single gRNAs 135, 115 and 117 indicated were cloned into Spectinomycin resistant Yamamoto B1 (Addgene # 58778), B2 (Addgene # 58779), B3 (Addgene # 58780) plasmids respectively using BbsI digestion (Supplementary Table 4). The multiplexed assembly is shown in (i). (i) Empty bridge control plasmid. Multiplexed plasmid with gRNAs 129, 135, 115, 117 (Supplementary Table 5). No soluble CRY2 Bridge is expressed from this plasmid, so it is referred to as the “Empty bridge control” plasmid. This plasmid was used in the “Empty bridge control” condition in Figure 1, 5 and Supplementary Figures 5, 13. (j) CRY2olig-mCherry plasmid with BbsI sites mutated. Plasmid used to PCR amplify the mCherry cassette for use in gRNA cloning. The original CRY2olig plasmid Addgene #60032 was mutated at two BbsI sites to give the CRY2olig-mCherry-mut2–1. Kanamycin resistant. (k) Empty target control plasmid. Soluble CRY2 (CRY2HA-2A-mCherry) cassette cloned into the plasmid S13.1. This plasmid was also used to facilitate insertion of CRY2 into the multiplexed plasmids without soluble CRY2. Schematic shown in Figure 1c. As this plasmid lacks the gRNA expression module it is referred to as the “Empty target control” plasmid and was used in Figures 1, 35 and Supplementary Figure 4, 611 and 13. (l) LADL Bridge + Target plasmid. Multiplexed plasmid with soluble CRY2 and gRNAs 129, 135, 115, 117 all targeting desert regions (Supplementary Table 6). Schematic shown in Figure 1c and plasmid used in Figures 15 and Supplementary Figures 1, 511 and 13. (m) LADL Bridge + Promoter Only Target plasmid. Multiplexed plasmid with gRNAs 115 and 117 targeting the Zfp462 promoter region with soluble CRY2 (Supplementary Table 6). This plasmid was used in the “One-sided guide control” condition in Supplementary Figures 710.

Supplementary Figure 3 Light box validation.

(a) Schematic of the CRY2olig-mCherry (Addgene #60032). (b) Circuit diagram of the light box (c) Experimental setup used to test CRY2 clustering using a customized light box. (d) Fluorescence imaging with a Texas Red filter to visualize CRY2HA-2A-mCherry. Scale bars, 10 µm. Images are representative of 2 independent experiments.

Supplementary Figure 4 Optimizing co-transfection efficiency of LADL constructs by testing different molar ratios and DNA masses.

(a) The LADL Anchor and Empty target plasmids were co-transfected with 1.5 µg, 2.0 µg or 2.5 µg of total mass at a 1:1 molar ratio, and imaged using phase contrast and fluorescence imaging in the TXR channel at 24 h post puromycin treatment. (b) The LADL Anchor and Empty target plasmids were co-transfected with 2.0 µg of total mass at 1:1, 2:1 and 4:1 molar ratio, and imaged as in (a). Scale bars, 500 µm. Images are representative of 2 independent experiments.

Supplementary Figure 5 Characterization of mouse embryonic stem (ES) cells after co-transfection with LADL constructs and exposure to blue light.

Images of mouse ES cells transfected with either the Empty anchor control, the Empty bridge control or the LADL (Anchor + Bridge + Target) combinations (Supplementary Figure 1) and imaged using phase contrast and fluorescence imaging with Texas Red filter to visualize CRY2HA-2A-mCherry at 0, 24 and 36 h post puromycin treatment. The Empty bridge samples, which should not have any mCherry signal, were used to set the acquisition parameters for the Texas Red channel. Scale bars, 500 µm. Images are representative of > 10 independent experiments.

Supplementary Figure 6 Chromosome-Conformation-Capture-Carbon-Copy (5C) global heatmaps at the larger Zfp462 and Klf4 gene loci in LADL-engineered mouse embryonic stem (ES) cells compared to relevant controls.

Heatmaps represent 5C signals in relative interaction frequency for an 804 kb genomic region around the Klf4 and Zfp462 genes (chr4:54,799,136–55,603,136) from mouse ES cells co-transfected with LADL (Anchor + Bridge + Target) constructs after 24 h blue light illumination at 5 mW/cm2 intensity (left column) and compared with two relevant controls: LADL–engineered cells in dark (middle column) and Empty target control in dark (right column). Zoom regions for engineered de novo loop (Box 1) and Klf4-stretch enhancer (SE) loop (Box 2) are shown in green boxes.

Supplementary Figure 7 5C heatmaps and boxplots of the engineered de novo loop after 24 h of blue light illumination in independent experiments.

(a-e) Zoomed-in 5C heatmaps represent engineered de novo loop between the targeted Zfp462 promoter (chr4:54,911,136–54,983,136; vertical) and pluripotency-specific Klf4 SE (chr4:55,451,136–55,523,136; horizontal) regions in LADL-engineered mouse ES cells after 24 h of (a, c-e) 5 mW/cm2 or (b) 1.5 mW/cm2 intensity blue light illumination, or in dark, from independent replicate experiments. (a-e) The loop is represented in relative interaction frequencies (top rows) and distance-corrected interaction scores (bottom rows). (a, c-d) Empty target controls (Anchor + Bridge) in dark are included in replicate 1, replicates 3–4. (b) Empty anchor control (Bridge + Target) in replicate 2 and (c) one-sided guide control (Anchor + Bridge + Zfp462 promoter only Target) in replicate 3 were exposed to the blue light for 24 h, or were in dark. See Supplementary Figure 1 for details of the plasmid combinations used. (f) Boxplots of relative interaction frequencies in the de novo engineered loop pixels (green box in a-e) from each independent experiment, respectively. Boxplots: central tendency = median, box minima = 25th percentile, box maxima = 75th percentile, notches = 95% confidence interval, whiskers = 1.5x interquartile range.

Supplementary Figure 8 Looping efficiency analysis highlights the engineered de novo loop from both gRNA anchors after blue light illumination.

(a) Zoomed-in 5C heatmaps representing the engineered de novo loop between the gRNA-targeted Zfp462 promoter (chr4:54,911,136–54,983,136; vertical) and pluripotency-specific Klf4 SE (chr4:55,451,136–55,523,136; horizontal). Zoom corresponds to Box 1 in the global heatmaps (see Figure 3a, b, Supplementary Figure 6). The brown box represents the 4C viewpoint at the Zfp462 promoter in (b–d), and Figure 3d. The blue box indicates the 4C viewpoint at Klf4 SE in (c, d left) and Figure 3e. The orange box represents the anchor point at the Klf4 SE used in (d right). See Supplementary Figure 1 for details of the plasmid combinations used. (b, c) Classic 4C looping efficiency plots. The line graph represents normalized interaction frequency of (b) the Zfp462 promoter anchor (brown vertical line) or (c) the SE anchor (brown vertical line) for five biological replicates in LADL + blue light (blue line), LADL + dark (black line) and additional controls in each replicate as listed: Empty target control in dark (grey line) is included in replicates1, 3 and 4; Empty anchor control + dark (grey line) and + blue light (light blue line) are included in replicate 2; and one-sided guide control + dark (light gray line) and + blue light (light blue line) is included in replicate 3. The single pixel of interest (engineered de novo loop) at (b) the targeted site in Klf4 SE and (c) the Zfp462 promoter are shaded in yellow. (d) Plots show the percent looping efficiency of the SE interactions at the Zfp462 promoter for each condition in all five replicates (left) after 24 h (intersection of brown and blue boxes in panel (a)) and (right) 4 h (intersection of brown and orange boxes in panel (a)) of blue light illumination. Red bars indicate medians of each condition. P-values computed using the unpaired two-sided Mann Whitney U test.

Supplementary Figure 9 5C heatmaps and boxplots of the endogenous loop between the pluripotency-specific Klf4 stretch enhancer (SE) and the Klf4 gene after 24 h of blue light illumination in independent experiments.

(a) Zoomed-in 5C heatmaps represent Klf4-SE loop in LADL-engineered cells after 24 h of 5 mW/cm2 blue light illumination, or in dark. (b) Klf4-SE loop in one additional replicate using 1.5 mW/cm2 and (c–e) three additional replicates using 5 mW/cm2 blue light illumination for 24 h. (a, c, d) Empty target controls (Anchor + Bridge) in dark are included in replicates 1, 3–4. (b) Empty anchor control (Bridge + Target) in replicate 2 and (c) one-sided guide control (Anchor + Bridge + Zfp462 promoter only Target) in replicate 3 were exposed to 1.5 mW/cm2 or 5 mW/cm2 blue light for 24 h, respectively, or were in dark. See Supplementary Figure 1 for details of the plasmid combinations used. (a–e) Klf4-SE loop is represented in relative interaction frequencies (top rows) and distance-corrected interaction scores (a-b bottom rows). (f) Boxplots of relative interaction frequencies in the Klf4-SE loop pixels (green box in a-e) after 24 h of blue light illumination from each independent experiment, respectively. Boxplots: central tendency = median, box minima = 25th percentile, box maxima = 75th percentile, notches = 95% confidence interval, whiskers = 1.5x interquartile range.

Supplementary Figure 10 Looping efficiency analysis highlights the effect on the endogenous Klf4-SE loop after engineering a loop with 24h blue light illumination.

(a) Zoomed-in 5C heatmaps representing the endogenous loop between pluripotency-specific Klf4 SE and Klf4 gene (chr4:55,447,136–55,583,136). The blue box represents the 4C viewpoint at Klf4 promoter in (b-c) and Figure 3g. The green box indicates the 4C viewpoint at Klf4 SE in (b, c) and Figure 3g. See Supplementary Figure 1 for details of the plasmid combinations used. (b) Classic 4C looping efficiency plots. The line graphs represent normalized interaction frequency of the Klf4 promoter (brown vertical line) for five biological replicates in LADL + blue light (blue line), LADL + dark (black line) and additional controls in each replicate as listed: Empty target control in dark (grey line) included in replicates 1, 3 and 4; Empty anchor control + dark (grey line) and + blue light (light blue line) included in replicate 2; and one-sided guide control +dark (light gray line) and + blue light (light blue line) included in replicate 3. The single pixel of interest (endogenous Klf4-SE loop) at the SE is shaded in yellow. (c) Strip charts show the % looping efficiency of the (left) Klf4 promoter interactions at the SE and (right) SE interactions at Klf4 promoter for each condition in all five replicates after 24h of blue light illumination. Values derived from the pixel at the intersection of green and blue boxes as shown in panel (a). Red bars indicate medians of each condition.

Supplementary Figure 11 5C heatmaps of the looping interactions between the Zfp462 gene and its pluripotency-specific enhancers (E1-E4) after 24 h illumination with 5 mW/cm2 intensity blue light.

(a-b) LADL-engineered mouse embryonic stem cells after 5 mW/cm2 blue light illumination (left column) after 24 h were compared with LADL-engineered cells in dark (middle column) and Empty target control in dark (right column). Heatmaps represent the regions around Zfp462 gene and its four pluripotency-specific enhancers E1, E2, E3 and E4 in chr4:54,859,136–55,351,136. (a) Relative interaction frequency and (b) distance-corrected interaction score heatmaps.

Supplementary Figure 12 Schematic of LADL experimental design with 4 and 24 h of blue light illumination.

Mouse embryonic stem cells were co-transfected with LADL (Anchor + Bridge + Target) plasmids or Empty target control (Anchor + Bridge only) plasmids. At the time of harvesting, the cells have undergone puromycin selection for at least 36 h in either 4 or 24 h of blue light exposure.

Supplementary Figure 13 RNA-FISH analysis of additional independent experiments.

LADL-engineered mouse embryonic stem cells and three other controls (LADL + dark, Empty target control + dark, Empty bridge control+dark) were exposed to 5 mW/cm2 blue light for 24 h in two independent replicate experiments. See Supplementary Figure 1 for details of the plasmid combinations used. (a–d) Strip charts representing (a, c) the number of total mRNA transcripts per cell and (b, d) the estimated level of nascent transcripts per allele for Zfp462 (upper row) and Klf4 (lower row) for two additional independent experiments. Red bars indicate means of each condition. (e, f) Histograms represent the proportion of cells with a specific number of actively expressing Zfp462 alleles in two additional biological replicates. (a, c, e, f) n = the number of cells, or (b, d) the number of active transcription alleles per condition. P-values computed using the unpaired one-tailed Mann Whitney U test.

Supplementary information

Supplementary Information

Supplementary Figs. 1–13 and Supplementary Tables 2–7 and 9

Reporting Summary

Supplementary Table 1

Primers used to clone LADL plasmids.

Supplementary Table 8

Summary of external sequencing libraries analyzed in this study.

Supplementary Table 10

5C primer sequences.

Supplementary Table 11

5C primer genomic coordinates.

Supplementary Table 12

Summary of mapped 5C sequencing reads.

Supplementary Table 13

Fluorescence-labeled oligonucleotide sequences for RNA–FISH.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Further reading

Fig. 1: Concept, design and implementation of the LADL system.
Fig. 2: Chromatin binding of LADL anchor (dCas9-CIBN) at the engineered sites increases after blue light exposure.
Fig. 3: LADL redirects a long-range interaction between an SE and a new target gene on blue light illumination.
Fig. 4: The LADL-engineered long-range interaction occurs as early as 4 h after blue light illumination.
Fig. 5: Functional effect of the LADL-engineered de novo interaction on endogenous gene expression.
Supplementary Figure 1: Overview of LADL components.
Supplementary Figure 2: Complete list of intermediate and final LADL plasmids.
Supplementary Figure 3: Light box validation.
Supplementary Figure 4: Optimizing co-transfection efficiency of LADL constructs by testing different molar ratios and DNA masses.
Supplementary Figure 5: Characterization of mouse embryonic stem (ES) cells after co-transfection with LADL constructs and exposure to blue light.
Supplementary Figure 6: Chromosome-Conformation-Capture-Carbon-Copy (5C) global heatmaps at the larger Zfp462 and Klf4 gene loci in LADL-engineered mouse embryonic stem (ES) cells compared to relevant controls.
Supplementary Figure 7: 5C heatmaps and boxplots of the engineered de novo loop after 24 h of blue light illumination in independent experiments.
Supplementary Figure 8: Looping efficiency analysis highlights the engineered de novo loop from both gRNA anchors after blue light illumination.
Supplementary Figure 9: 5C heatmaps and boxplots of the endogenous loop between the pluripotency-specific Klf4 stretch enhancer (SE) and the Klf4 gene after 24 h of blue light illumination in independent experiments.
Supplementary Figure 10: Looping efficiency analysis highlights the effect on the endogenous Klf4-SE loop after engineering a loop with 24h blue light illumination.
Supplementary Figure 11: 5C heatmaps of the looping interactions between the Zfp462 gene and its pluripotency-specific enhancers (E1-E4) after 24 h illumination with 5 mW/cm2 intensity blue light.
Supplementary Figure 12: Schematic of LADL experimental design with 4 and 24 h of blue light illumination.
Supplementary Figure 13: RNA-FISH analysis of additional independent experiments.