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