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. 2022 Oct 10;41(4):488–499. doi: 10.1038/s41587-022-01494-w

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a, Schematic of mini PRA. A pool of reporter plasmids with varied synthetic enhancers containing TetO sites is integrated into the landing pad by the LSR, selected for using puromycin, and then reporter activation is induced using doxycycline, which causes rTetR-VP48 to bind TetO. Highly activated and lowly activated cells are magnetically separated; the enhancers are sequenced from gDNA in each cell population; and a ratio of reads is computed as a measurement of enhancer strength. b, Individual enhancer reporters with a varied number of TetO transcription factor binding sites were integrated into the AAVS1 safe harbor by HDR or into the landing pad using the Kp03 LSR. Flow cytometry measurements were taken 2 days after induction with doxycycline. Due to varied voltage settings on the cytometer, the x-axes are not comparable in absolute terms (n = 1 cell line replicate, and a second replicate is shown in Supplementary Fig. 3a). c, A small pooled library of synthetic enhancer reporters was integrated into the AAVS1 safe harbor by HDR or a clonal landing pad by the Kp03 LSR and measured by separation and sequencing (n = 2 integration replicates for HDR; n = 3 integration replicates for LSR; dots show the mean; error = s.d.). ρ is the Spearman correlation between the PRA measurement of enhancer strength and the number of TetO sites in the enhancer. For the LSR, pooled measurements (left y-axis, red circles) correlate with the percentage of citrine⁺ cells from individual reporter assays (right y-axis, black x, Pearson’s r = 0.94). d, Schematic for a cloning-free strategy to install libraries. A linear dsDNA library of elements containing the attP site is generated by PCR and directly delivered to landing pad cells. e, Schematic of an amplicon library generated by PCR from an attP-mCherry-pA template, where the reverse primer contains a 6×N barcode. f, Distribution of barcodes in the initial amplicon libraries (read depths 216–272×) and in gDNA extracted from cells 7 days after electroporation with 750 ng of amplicon (read depths 290–357×). dox, doxycycline.