ef1a neurod1 p2a hygro barcode  (Addgene inc)

Journal: bioRxiv

Article Title: Systematic mapping of enhancers, silencers, and topological elements with prime editing deletion screens

doi: 10.64898/2026.10.08.756856

Figure Lengend Snippet: A. Schematic of the Swap-seq workflow for measuring the effects of DNA sequence deletions on gene expression. Each Swap-seq edit uses a pair of pegRNAs to delete a 50- to 250-bp target sequence and replace it with a barcode cassette containing an edit-specific 8-bp barcode together with sequences common to all edits, including PCR primer-binding sites (yellow) and a 3′ overlap sequence (purple). In a Swap-seq screen, hundreds of such edits are designed to perturb sequences surrounding a gene of interest. Cells are infected with a lentiviral pegRNA-pair library at low multiplicity of infection (MOI); after 14 days of editing, cells are cultured without doxycycline for 7 days before undergoing FlowFISH sorting and quantification of changes in gene expression. B. Individual Swap-seq editing test in three regions: the PPIF promoter, a candidate enhancer with high accessibility and H3K27ac signal (enhancer e2 in G ), and a region with low accessibility (hg19: chr10:81,069,000-81,069,517). Error bars: 95% CI for the mean. C. Correlation of effect sizes between two biological replicates, after removing edits that did not pass the 0.001% frequency threshold. Effects for each edit are averaged among barcodes. (Pearson’s r = 0.97, n = 117 edits). D. Editing efficiency and number of cells assayed for each barcode. A barcode passed the threshold in a given technical replicate if aligned reads exceeded 0.001% of all reads. Bottom axis, mean allele frequency calculated as the percent of reads aligned to the barcode averaged across replicates; y-axis, number of barcodes; top axis, number of cells assayed. E. Volcano plot of Swap-seq edits passing the frequency threshold. Edits were considered significant at BH-corrected p < 0.001 (horizontal dashed line). Error bars: 95% CI for the mean. F. Swap-seq screen candidate elements at the PPIF locus. In total, 24 candidate regulatory elements were targeted and the PPIF promoter was included as a positive control. *Two elements with no edits above the frequency threshold of 0.001%. G. Swap-seq effects on PPIF expression. Red, significant edits (BH-corrected p < 0.001, one-sample t-test); gray, non-significant edits. H. Swap-seq edits spanning the promoter, splice junction, and enhancer g1. Red, significant edits (BH-corrected p < 0.001, one-sample t-test); gray, non-significant edits.

Article Snippet: Following extraction, we split genomic DNA into PCR replicates and amplified the Swap-seq barcode cassettes with Q5 High-Fidelity 2X Master Mix (New England Biolabs #M0492L) and 0.5 uM of each amplification primer ( table S1 ) with the following program: 98°C for 1 minute and then 26 cycles of 98°C for 10 seconds, 69°C for 20 seconds, 72°C for 20 seconds, followed by a final 72°C extension for 2 minutes.

Techniques: Sequencing, Gene Expression, Binding Assay, Infection, Cell Culture, Positive Control, Expressing, Polymerase Chain Reaction, ChIP-sequencing

Journal: bioRxiv

Article Title: Systematic mapping of enhancers, silencers, and topological elements with prime editing deletion screens

doi: 10.64898/2026.10.08.756856

Figure Lengend Snippet: A. Comparison of regulatory elements identified by Swap-seq and CRISPRi. An element was classified as significant in Swap-seq if at least one edit within the element was significant (one-sample t-test, BH-corrected p < 0.001). CRISPRi used the average effects of all gRNAs targeting the same region (one-sample t-test, BH-corrected p < 0.05). Elements were grouped according to the hypothesized mechanisms based on their candidate element categories and effects on PPIF gene expression. B. Correlation between Swap-seq and CRISPRi effect sizes. Elements within the PPIF gene body, which are not testable by CRISPRi, were excluded from this analysis; when an element contains several significant edits, the one with the strongest effect was used for comparison with CRISPRi (Pearson’s r = 0.75, n = 18 elements). Error bars: 95% CI for the mean. C. Swap-seq edits overlap key transcription factor binding sites (TFBS) within regulatory elements. Close-up views of four elements with significant effects on PPIF expression (left to right: e2, a PPIF enhancer with high H3K27ac; e5, a low-H3K27ac enhancer; g1, an enhancer in intron 1 of PPIF ; c2, a CTCF binding site). ChromBPNet attribution scores are plotted together with TF-MoDISco motif annotations. All edits that passed the frequency threshold are plotted with width corresponding to the deleted region. Significance determined by one-sample t-test: red, significant edits (BH-corrected p < 0.001); gray, non-significant edits. Error bars: 95% CI for the mean. D. Schematic of the downstream lentiMPRA. Candidate regulatory elements were cloned downstream of the promoter and an ORF–barcode–poly(A) cassette (ORF-BC-pA). Cells were infected with the lentiviral library at high MOI, and barcodes were sequenced from both DNA and RNA; CRE activity was calculated as the ratio of RNA to DNA barcode abundance. E. LentiMPRA validation of candidate enhancers. Each dot represents a barcode from two biological replicates. A sequence lacking predicted TFBS (noTFBS) served as a negative control for basal promoter strength. Significance determined by two-sample t-test: *p < 0.05, **p < 0.01, ***p < 0.001. Error bars: 95% CI for the mean. F. CTCF deletion alters 3D genome contacts at the PPIF locus. Contact frequencies were averaged across region-capture Micro-C (RCMC) measurements from three independent clones, and log2 fold-change was calculated as log2(c2-deleted clones / WT clones) at 1.6 kb bin sizes. G. Quantification of contact changes associated with CTCF deletion, with the viewpoint on the PPIF promoter. Bars show changes in contact frequency between the PPIF promoter and each regulatory element, averaged across three clonal lines at 1.6 kb binsize. Significance determined by two-sample t-test: *p < 0.05, **p < 0.01, ***p < 0.001. Error bars: 95% CI for the mean.

Article Snippet: Following extraction, we split genomic DNA into PCR replicates and amplified the Swap-seq barcode cassettes with Q5 High-Fidelity 2X Master Mix (New England Biolabs #M0492L) and 0.5 uM of each amplification primer ( table S1 ) with the following program: 98°C for 1 minute and then 26 cycles of 98°C for 10 seconds, 69°C for 20 seconds, 72°C for 20 seconds, followed by a final 72°C extension for 2 minutes.

Techniques: Comparison, Gene Expression, Analysis, Binding Assay, Expressing, Clone Assay, Infection, Activity Assay, Biomarker Discovery, Sequencing, Negative Control