Journal: bioRxiv

Article Title: S-SELeCT: A Human-Evolved Serine Integrase System for Efficient Large-Cargo Genome Integration

doi: 10.64898/2026.01.30.702954

Figure Lengend Snippet: Inversion assay & screen ( A ) To minimize false-positive signals, mutant integrases are subjected to a three-exon GFP plasmid inversion test. If no recombination occurs, the central GFP exon remains in the reverse orientation, which prevents production of green fluorescence above background. In cells with an active variant, the two attachment sites are recombined, which leads to inversion of exon 2 and the production of complete GFP. ( B ) Site A and C31 attP were divided into 5 segments, A1-A5 ( – ). Intermediate sites are named after the segment of C31 attP that has been mutated in both half sites to match the respective Site A sequence. ( C ) Variants with improved activity on A2 intermediate. WTxA2 and WTxWT show the activity of WT C31-int in the A2 and WT attP x attB inversion assays, respectively. Assay performed for 72 hours in HEK293 cells that stably express the respective variant from the H11 locus. ( D ) Variants with improved activity on A4 intermediate. WTxA4 and WTxWT show the activity of WT C31-int in the A4 and WT attP x attB inversion assays, respectively. Assay performed for 72 hours in HEK293 cells that stably express the respective variant from the H11 locus. ( E) Five variants with the strongest ability to recombine the attachment sites of interest were tested using the plasmid inversion assay over 96 hours in HEK293 cells that stably express the respective variant from the H11 locus. For (C), (D) and (E), a split-intein mCherry system was used to limit analysis to cells that both received the inversion plasmid and that also expressed the variant integrase (single copy expressed from H11 locus). For plots with error-bars (standard error; STDEV/SQRT), N=3 biological replicates.

Article Snippet: When cells were seeded at a higher cell density (HCD; 200k in 24 ww), we sequenced 3-4 technical replicates derived from two biological replicates of ratio E (4 tech. reps. from first, 3 tech. reps. from second), and 3 technical replicates derived from one biological sample of ratio F. Sequencing was performed both in-house using a MinION device, and via the Plasmidsaurus custom sequencing service at a 111 megabase scale per sample.

Techniques: Mutagenesis, Plasmid Preparation, Fluorescence, Variant Assay, Sequencing, Activity Assay, Stable Transfection

Journal: bioRxiv

Article Title: S-SELeCT: A Human-Evolved Serine Integrase System for Efficient Large-Cargo Genome Integration

doi: 10.64898/2026.01.30.702954

Figure Lengend Snippet: Localization optimization ( A ) Split-GFP site A integration efficiency assay. Wildtype C31 attP was placed at both site A loci in HEK293 cells. Downstream of each attP, a splice acceptor, 3’ segment of GFP, and transcription-termination sequence were also introduced. To enable detection of site-specific integration, a donor plasmid was constructed that contains the elements needed to form a complete GFP-expression cassette: CMV promoter, 5’ GFP segment, splice donor, wildtype attB site. After co-transfection of the donor and integrase-expression plasmids, cells where site-specific integration has occurred can be identified by looking for green fluorescence. Integration can happen at one (panels i and ii) or both loci (panel iii). ( B ) WT C31-int dMad7 fusion protein expression cassette used to test impact of different gRNA on site A localization efficiency. A 5’ mCherry segment fused to a trans-splicing intein domain was co-expressed and separated from int-dMad7 via a 2A-skipping peptide. In the donor plasmid, the remaining 3’ mCherry segment fused to the complementary trans-splicing intein domain was co-expressed with the 5’ GFP segment mentioned in (A), and these two proteins were separated via 2A peptide ribosome skipping. ( C ) Results of site A localization experiments for the indicated guide RNAs and combinations, in transfected cells that express the fusion protein most strongly. Assay was performed for 72 hours in HEK293 cells that stably express the fusion protein from the H11 locus. ( D ) Expression (mCherry) and GFP gating. The rightmost mCherry gate was used to analyze the cells summarized in (C).

Article Snippet: When cells were seeded at a higher cell density (HCD; 200k in 24 ww), we sequenced 3-4 technical replicates derived from two biological replicates of ratio E (4 tech. reps. from first, 3 tech. reps. from second), and 3 technical replicates derived from one biological sample of ratio F. Sequencing was performed both in-house using a MinION device, and via the Plasmidsaurus custom sequencing service at a 111 megabase scale per sample.

Techniques: Sequencing, Plasmid Preparation, Construct, Expressing, Cotransfection, Fluorescence, Transfection, Stable Transfection

Journal: bioRxiv

Article Title: S-SELeCT: A Human-Evolved Serine Integrase System for Efficient Large-Cargo Genome Integration

doi: 10.64898/2026.01.30.702954

Figure Lengend Snippet: Minichromosome assay ( A ) Site A-splice acceptor-3’ GFP-polyA was cloned in a plasmid with 2.5 - 5 kb Site A genomic sequence on each side, along with other elements needed for EBNA1-mediated extrachromosomal maintenance in human cells (EBNA1 expression, oriP, puromycin-resistance marker). The 20 kb (SAR MAX, not shown) and 15 kb (SAR 1) plasmids have 5 kb and 2.5 kb genomic Site A sequence on each side (10 and 5 kb total), respectively. ( B ) When the mini-chromosome assay was used in screens, we used the depicted alternative-splicing expression construct to produce the needed solo and fused forms of each variant integrase. ( C ) Results from individual tests of the top-performing variants in the mini-chromosome assay. Assay was performed for 72 hours in HEK293 cells with the Site A SAR1 mini-chromosome that stably expressed the indicated variant using an alternative-splicing cassette from the H11 locus. N=3 biological replicates, error bars are standard error (STDEV/SQRT).

Article Snippet: When cells were seeded at a higher cell density (HCD; 200k in 24 ww), we sequenced 3-4 technical replicates derived from two biological replicates of ratio E (4 tech. reps. from first, 3 tech. reps. from second), and 3 technical replicates derived from one biological sample of ratio F. Sequencing was performed both in-house using a MinION device, and via the Plasmidsaurus custom sequencing service at a 111 megabase scale per sample.

Techniques: Clone Assay, Plasmid Preparation, Sequencing, Expressing, Marker, Alternative Splicing, Construct, Variant Assay, Stable Transfection

Journal: bioRxiv

Article Title: BAF complex-independent gene activation by SS18::SSX

doi: 10.64898/2026.01.26.701739

Figure Lengend Snippet: (A) RNA-seq heatmap showing the 1,000 most variable genes with a cutoff z-score of 4 from HSSY-II cells treated for 72h with DMSO or 500nM ACBI1 or of HSSY-II-Cas9 cells expressing a safe sgRNA as control or with double guides targeting SSX + a control region (sgSSX/SAFE), SMARCA2 + SMARCA4 (sgSMARCA2/4) or SMARCC1 + SMARCC2 (sgSMARCC1/2). n=2 biological replicates. (B) Log2-transformed fold change of FPKM values in HSSY-II RNA-seq relative to DMSO or sgSAFE over selected SS18::SSX target genes. Data represents the mean. (C) qRT-PCR displaying log2-transformed fold change of mRNA levels normalised by GAPDH in Yamato-SS cells treated for 72h with DMSO or 500nM ACBI1 or of Yamato-SS Cas9 cells expressing sgSAFE, (sgSSX/SAFE), (sgSMARCA2/4) or (sgSMARCC1/2) relative to DMSO or sgSAFE for 7 days. Data represents the mean of n=2 biological replicates. (D) Left: heatmaps for SMARCA4 and SS18::SSX1 (endogenously HA tagged) ChIP-seq from Banito et al., 2018 over SMARCA4 peaks (n = 20,278). Rows correspond to ±5-kb regions across the midpoint of each SMARCA4-enriched region, ranked by increasing signal. Right: ATAC-seq heatmaps displaying log2-transformed fold change of ACBI1 treated cells over DMSO after 24h or 72h of treatment and of sgSSX/SAFE expressing cells over sgSAFE cells. n=2 biological replicates. (E) Scatter plot of log2-transformed fold change of FPKM values in HSSY-II RNA-seq relative to DMSO or sgSAFE over genes associated with SMARCA4 occupancy and showing <-1.5 fold down-regulation upon sgSSX/SAFE knockout. Biological replicates are combined using their mean. Bold line represents median and dotted line represents -1.5 cutoff. p-values represent two-tailed Wilcoxon test. (F) Gene tracks for SMARCA4, SS18::SSX1 ChIP-seq, Log2-Fold changes of ATAC-seq or Log2-Fold changes of RNA-seq at loci from (E) such as MAFA , MNX1 , SKOR1 , FGF8 , WNT7B , BMP7 and ONECUT3 .

Article Snippet: RNA sequencing of SSX chimeric fusions in figure (6F, H, I) was done using Plasmidsaurus RNA sequencing service.

Techniques: RNA Sequencing, Expressing, Control, Transformation Assay, Quantitative RT-PCR, ChIP-sequencing, Knock-Out, Two Tailed Test

Journal: bioRxiv

Article Title: BAF complex-independent gene activation by SS18::SSX

doi: 10.64898/2026.01.26.701739

Figure Lengend Snippet: (A) Schematics showing timeline for ACBI1/DMSO treatment and SS18::SSX/EV delivery in KHOS-240S cells. (B) Western Blot of KHOS-240S whole cell extracts after 24h pre-treatment with DMSO or 500nM ACBI1 (Day 0), or at day 2 and 3 following expression of an empty vector (EV) or SS18::SSX1 transgene and continued treatment with DMSO or ACBI1. (C) RNA-seq heatmap showing the 1,000 most variable genes with a cutoff z-score of 2 from KHOS-240S cells pre-treated with DMSO or 500nM ACBI1 for 24h prior to expression of an empty vector (EV) or SS18::SSX1 transgene for additional 72h and co-treatment with DMSO or ACBI1. n=2 biological replicates. Dotted square highlights genes up- regulated by SS18::SSX1 expression (D) Log2-transformed fold change of FPKM values in KHOS-240S RNA-seq relative to EV/DMSO over selected SS18::SSX target genes. Data represents the mean. (E) Scatter plot of log2-transformed fold change of FPKM values in KHOS-240S RNA-seq relative to EV/DMSO over genes up-regulated by SS18::SSX1 expression and showing >1.5 fold up-regulation. Biological replicates are combined using their mean. Bold line represents median and dotted line represents 1.5 cutoff. p-values represent two-tailed Wilcoxon test. (F) Left: heatmaps for SS18::SSX1 (HA tagged-transgene) CUT&RUN from Benabdallah et al., 2023 over SS18::SSX1 peaks (n = 16,687). Rows correspond to ±5-kb regions across the midpoint of each SS18::SSX1-enriched region, ranked by increasing signal. Right: ATAC-seq heatmaps (in greys) of KHOS-240S cells pre-treated with DMSO or 500nM ACBI1 for 24h prior to expression of an empty vector (EV) or SS18::SSX1 transgene for additional 72h and co-treatment with DMSO or ACBI1 or ATAC-seq heatmaps (in blue-red gradient) of comparison using log2- transformed fold change of either ACBI1 relative to DMSO in EV or SS18::SSX1 condition or SS18::SSX relative to EV in DMSO or ACBI1 conditions. n-2 biological replicates. (G) Gene tracks for SS18::SSX1 CUT&RUN, ATAC-seq of KHOS-240S cells pre-treated with DMSO or 500nM ACBI1 for 24h prior to expression of an empty vector (EV) or SS18::SSX1 transgene for additional 72h and co-treatment with DMSO or ACBI1 or Log2-Fold changes of RNA-seq of SS18::SSX relative to EV in DMSO or ACBI1 conditions at SS18::SSX1 target loci in KHOS-240S cells displaying up-regulation upon SS18::SSX transgene expression such as IGF2 , TPPP , MAFA , SHH , HES4 and ONECUT3 .

Article Snippet: RNA sequencing of SSX chimeric fusions in figure (6F, H, I) was done using Plasmidsaurus RNA sequencing service.

Techniques: Western Blot, Expressing, Plasmid Preparation, RNA Sequencing, Transformation Assay, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: BAF complex-independent gene activation by SS18::SSX

doi: 10.64898/2026.01.26.701739

Figure Lengend Snippet: (A) Schematic of SS18::SSX displaying SS18 (orange) main domains, SS18 N-terminal homology (SNH, purple) and Glutamine, Proline, Gylcine, Tyrosine rich domain (QPGY, green). (B) Schematic of eGFP-fused constructs, eGFP alone, wild-type SS18::SSX isoform 2 (FL), SS18::SSX lacking SNH domain (ΔSNH) or SS18::SSX lacking QPGY domain (ΔQPGY). (C) Co-immunoprecipitation (co-IP) pulling down on eGFP in KHOS-240S cells expressing eGFP constructs representing one replicate. (D), (E) Volcano plots of mass spectrometry data following eGFP pull down of ΔSNH (E) or ΔQPGY (F) enrichment normalised to SS18 wild-type (FL). Data represents log2 fold change enrichment plotted on the x-axis and -log10 (adjusted p value) plotted on the y-axis. Blue/Red dots indicate lost/gained interactors relative to SS18::SSX1 isoform 2 (FL). Black dots indicate BAF members. n = 4 biological replicates. (F) List of BAF members displaying decreased interaction and their associated log2 fold change value in ΔSNH (left) or ΔQPGY (right) pull downs normalised to wild-type SS18::SSX1 isoform 2 (FL). Listed members are shown as black dots in (D) and (E). (G) RNA-seq heatmap showing the 1,000 most variable genes with a cutoff z-score of 4 from KHOS-240S cells expressing eGFP, FL, ΔSNH or ΔQPGY. n = 2 biological replicates. (H) Log2-transformed fold change of FPKM values in KHOS-240S RNA-seq relative to eGFP over selected SS18::SSX target genes. Data represents the mean. (I) Scatter plot of log2-transformed fold change of FPKM values in KHOS-240S RNA-seq relative to eGFP over genes up-regulated by SS18::SSX1 expression and showing >2 fold up-regulation. Biological replicates are combined using their mean. Bold line represents median and dotted line represents 2 cutoff. (J) Log2 fold change plot between ΔSNH and ΔQPGY mass spectrometry data following eGFP pull down in KHOS-240S cells. (K) Volcano plot of miniTurbo-v5-SS18::SSX2 proximity proteomics in HEK293T cells (miniTurbo = mT). Data represents log2 fold change enrichment plotted on the x-axis (mT-v5-SS18::SSX2 / mT-control)and -log10 (adjusted p value) plotted on the y-axis. Black dots indicate BAF members. EP300 and RBM14 are highlighted in green; mT and mT-SS18::SSX2 are highlighted in orange. n = 3 biological replicates. (L) Left: Immunofluorescence of HEK293T cells expressing eGFP, FL, ΔSNH or ΔQPGY (cyan) stained for EP300 (magenta). Images are representative of n = 2 biological replicates. Scale bar, 5 μm. Right: Profile of fluorescence intensity of eGFP, FL, ΔSNH or ΔQPGY over eGFP foci. Data represent the mean ± S.E.M of n = 2 biological replicates. For each replicate and condition, > 10 foci where profiled. For eGFP quantification in (N), random profiles across nucleus where generated. R² indicates Pearson correlation coefficient.

Article Snippet: RNA sequencing of SSX chimeric fusions in figure (6F, H, I) was done using Plasmidsaurus RNA sequencing service.

Techniques: Construct, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Mass Spectrometry, RNA Sequencing, Transformation Assay, Control, Immunofluorescence, Staining, Fluorescence, Generated

Journal: bioRxiv

Article Title: BAF complex-independent gene activation by SS18::SSX

doi: 10.64898/2026.01.26.701739

Figure Lengend Snippet: (A) Left: Immunofluorescence of HEK293T cells expressing eGFP or wild-type SS18::SSX1, either untreated (FL) (cyan) or treated for 72h with 500nM ACBI1 or 500nM dCBP-1 stained for EP300 (magenta). Images are representative of n = 2 biological replicates. Scale bar, 5 μm. Right: Profile of fluorescence intensity. Data represent the mean ± S.E.M of n = 2 biological replicates. For each replicate, > 10 foci where profiled. R² indicates Pearson correlation coefficient. (B ) Western Blot of KHOS-240S whole cell extracts treated for 24h with DMSO, 500nM or 1uM dCBP- 1 revealed using EP300, CREBBP or β-actin antibodies. (C) RNA-seq heatmap showing the 1,000 most variable genes with a cutoff z-score of 4 from KHOS-240S cells expressing eGFP or SS18::SSX1 (FL) treated for 72h with either DMSO, 500nM ACBI1 or 500nM dCBP-1. n = 2 biological replicates. ( D) Log2-transformed fold change of FPKM values in KHOS-240S RNA-seq relative to eGFP over selected SS18::SSX target genes. Data represents the mean. (E) Scatter plot of log2-transformed fold change of FPKM values in KHOS-240S RNA-seq relative to eGFP over genes up-regulated by SS18::SSX1 expression and showing >2 fold up-regulation. Biological replicates are combined using their mean. Bold line represents median and dotted line represents cutoff of 2. (F) RNA-seq heatmap showing the 1,000 most variable genes with a cutoff z-score of 4 from wild-type hMSC or cells expressing eGFP-SSX1, eGFP-SS18::SSX1 isoform1, eGFP-SS18::SSX1 isoform2, eGFP-EWSR1::SSX1 or eGFP- EPC1::SSX1 treated for 48h with 500nM dCBP-1. n = 2 biological replicates. (G) Top: Immunofluorescence of HEK293T cells expressing wild-type eGFP-SSX1, eGFP-SS18::SSX1 isoform1, eGFP-SS18::SSX1 isoform2, eGFP-EWSR1::SSX1 or eGFP-EPC1::SSX1(cyan) stained for EP300 (magenta). Images are representative of n = 2 biological replicates. Scale bar, 5 μm. Bottom: profile of fluorescence intensity of eGFP constructs over SSX-rich eGFP foci. Data represent the mean ± S.E.M of n = 2 biological replicates. For each replicate and condition, > 10 foci where profiled. R² and r indicate Pearson correlation coefficients. (H) Scatter plot of log2-transformed fold change of CPM values in hMSC RNA-seq relative to untreated hMSC over the union genes up-regulated by SS18::SSX1 isoform1 (log2FC >2) and up-regulated by EPC1::SSX1 (log2FC >1). Biological replicates are combined using their mean. Bold line represents median. (I) Log2-transformed fold change of CPM values in hMSC RNA-seq relative to untreated cells over selected SSX target genes. Data represents the mean. (J) qRT-PCR displaying log2- transformed fold change of mRNA levels normalised by GAPDH in HSSY-II, SYO-1, CME-1 and Yamato-SS cells treated for 72h with 500nM dCBP-1 relative to DMSO. Data represents the mean of n = 2 biological replicates. (K) Viability of 4 synovial sarcoma cell lines (Yamato-SS, SYO-1, CME-1, HSSY-II) and KHOS-240S osteosarcoma cells treated with various concentrations of dCBP-1. The symbols represent the mean ± S.E.M of n = 3 biological replicates, the solid line represent the nonlinear curve fitting and its associated IC50. p-value represents extra sum-of- squares F test between KHOS-240S and SYO-1 nonlinear regression curves.

Article Snippet: RNA sequencing of SSX chimeric fusions in figure (6F, H, I) was done using Plasmidsaurus RNA sequencing service.

Techniques: Immunofluorescence, Expressing, Staining, Fluorescence, Western Blot, RNA Sequencing, Transformation Assay, Construct, Quantitative RT-PCR

Journal: STAR Protocols

Article Title: Protocol to perform multiplexed assays of variant effect using curated loci prime editing

doi: 10.1016/j.xpro.2025.103851

Figure Lengend Snippet: Example of cliPE editing in HAP1 cells Amplicon sequencing was used to validate editing of HAP1 cells in the targeted region of TSC2 exon 17. Fastq files were aligned to the human hg38 reference and bam files were viewed in IGV software.

Article Snippet: LR sequencing services like Plasmidsaurus are useful for initial QC of an epegRNA library, but it is difficult to accurately estimate the frequency of each epegRNA within the pool with the limited number of reads you routinely receive.

Techniques: Amplification, Sequencing, Software