Journal: bioRxiv

Article Title: Site-specific integration of transposon via engineered piggyBac transposase

doi: 10.64898/2026.01.20.700476

Figure Lengend Snippet: (A) Illustration of the split-GFP reporter system to test site-specific integration by Cas9-PBase. With this reporter, the transposon donor expressing BFP, PuroR and N-terminal GFP is directed to the target site upstream of C-GFP by Cas9-PBase. BFP positive cell counts represent the transfection rate (Day 4) or total integrated rate (Day14). GFP positive cell counts represent forward on-target integration. (B) Site-specific integration efficiency mediated by selected Cas9-PBase mutants. The ratio of 2 × GFP/BFP(D4) represents the site-specific integration efficiency. Cas9-PBase R372A_K375A_D450N is used as the benchmark. (C) The on-target ratio of selected Cas9-PBase mutants. Stable integrated cells were enriched with puromycin and analyzed by flow cytometry at day 14. The on-target percentage is represented as 2×GFP/BFP(D14). All data was analyzed and presented as Mean ± SD of three biological replications, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Article Snippet: The Cas9-PBase fusion constructs were generated using hCas9_D10A (Addgene #41816) as the backbone, with the D10A nickase mutation reverted to wild-type D10 to restore full nuclease activity.

Techniques: Expressing, Transfection, Flow Cytometry

Journal: bioRxiv

Article Title: Site-specific integration of transposon via engineered piggyBac transposase

doi: 10.64898/2026.01.20.700476

Figure Lengend Snippet: (A) Two plasmids system to test the site-specific integration of piggyBac transposon at endogenous genomic loci. The transposon plasmid expresses NeoR & GFP. The enzyme plasmid expresses Cas9-PBase and transcribes sgRNA for target site. (B) Total integration efficiency of Cas9-PBase at endogenous genomic loci. Integration efficiency is represented by GFP(D14)/GFP(D3) in HeLa cells. The negative control (NC) is background integration without Cas9-PBase enzyme. (C) On-target clones at GSH1 locus determined by junction-PCR. Single cell clones were digested and lysed for junction PCR with primer pairs annealing to genomic loci and transposon donor respectively. On-target clones were determined with PCR positive bands. (D) Characteristics of junction sequences between ITR and genomic DNA. Representative sequencing reads of on-target clones are aligned to the reference sequence. Small insertions or deletions were found in the junction, suggesting the transposon sequence is integrated in HITI manner. Arrows indicate two primer pairs for junction PCR. All data was analyzed and presented as Mean ± SD of three biological replications, *p≤0.05, **p≤0.01, ***p≤0.001.

Article Snippet: The Cas9-PBase fusion constructs were generated using hCas9_D10A (Addgene #41816) as the backbone, with the D10A nickase mutation reverted to wild-type D10 to restore full nuclease activity.

Techniques: Plasmid Preparation, Negative Control, Clone Assay, Sequencing

Journal: bioRxiv

Article Title: Site-specific integration of transposon via engineered piggyBac transposase

doi: 10.64898/2026.01.20.700476

Figure Lengend Snippet: (A) Detailed interactions between N286/T557 of PBase with phosphates in the ITR DNA. (B) Flow cytometry analysis of further engineered Cas9-PBase in the split-GFP system on day 4 post transfection. Cas9-PBase N347A_R376E_D450N is termed as v1, Cas9-PBase N347A_R376E_D450N_T557R is v2, and Cas9-PBase N347A_R376E_D450N_N286R_T557R is v3. The negative control (NC) is background fluorescence of reporter cells. Representative image of n = 3. (C) Efficiency of site-specific integration by further engineered Cas9-PBase. The ratio of 2×GFP/BFP (D4) represents the site-specific integration efficiency. (D) Total integration efficiency of Cas9-PBase at endogenous genomic loci. Integration efficiency is represented by GFP(D14)/GFP(D3) in HEK293T cells. Background integration without Cas9-PBase has been deducted. (E) Schematic illustration of the sgRNA-Transposon plasmid with sgRNA target sequences located upstream of the 5’ ITR. (F) Efficiency of site-specific integration of sgRNA-Transposon by Cas9-PBase versions in the split-GFP system. The ratio of 2×GFP/BFP (D4) represents the site-specific integration efficiency. (G) Flow cytometry analysis of puromycin-enriched cells on day 14 post transfection. Stable integrated cells in were selected with puromycin. The negative control (NC) is background fluorescence without transfection. Representative image of n = 3. (H) On-target ratio of the sgRNA-Transposon and Transposon by Cas9-PBase variants. The ratio of 2×GFP/BFP (D14) in represents the on-target percentage. All data was analyzed and presented as Mean ± SD of three biological replications, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Article Snippet: The Cas9-PBase fusion constructs were generated using hCas9_D10A (Addgene #41816) as the backbone, with the D10A nickase mutation reverted to wild-type D10 to restore full nuclease activity.

Techniques: Flow Cytometry, Transfection, Negative Control, Fluorescence, Plasmid Preparation

Journal: bioRxiv

Article Title: Site-specific integration of transposon via engineered piggyBac transposase

doi: 10.64898/2026.01.20.700476

Figure Lengend Snippet: (A) Strategy of iPSC editing by Cas9-PBase. The sgRNA was designed to target exon 1 of β2M gene, allowing knock-out of β2M and knock-in of the 7.1 kb cargo within a single editing step. (B) Transgenes expression quantification by flow cytometry analysis of edited iPS cells. The edited #29 iPSC clone, the β2M -/- control clone and the wild-type iPSCs were stained with antibodies to detect expression of CD47, HLA-E and element 3. (C) Characterization of iNK cells. Differentiated iNK cells at day 28 of differentiation stage 2 were stained with CD45 and CD56 antibodies, followed by flow cytometry analysis. (D) Transgenes expression quantification by flow cytometry analysis of iNK cells. Differentiated iNK cells at day 28 of differentiation stage 2 were stained with antibodies to detect expression of CD47, HLA-E and element 3. (E) Cytotoxicity by allogeneic PBNK to iNK cells. Prelabeled iNK cells were co-cultivated with allogeneic PBNK cells at multiple E:T ratios for 6 hours. The cytotoxicity of PBNK to iNK cells was measured with SYTOX staining and flow cytometry analysis. Representative image of n = 3. All data was analyzed with Mean ± SD of three biological replications, *p≤0.05, **p≤0.01, ***p≤0.001.

Article Snippet: The Cas9-PBase fusion constructs were generated using hCas9_D10A (Addgene #41816) as the backbone, with the D10A nickase mutation reverted to wild-type D10 to restore full nuclease activity.

Techniques: Knock-Out, Knock-In, Expressing, Flow Cytometry, Control, Staining

Journal: bioRxiv

Article Title: Site-specific integration of transposon via engineered piggyBac transposase

doi: 10.64898/2026.01.20.700476

Figure Lengend Snippet: PBase in the fusion protein with high excision activity cleaves the transposon and binds to ITR sequences. Since mutated PBase has low integration activity, the transposon payload is not integrated at the “off-target” TTAA sites. Instead, it is directed to the target site by sgRNA, where Cas9 generates the DSB. The payload captured by Cas9-PBase fusion protein to the vicinity of DSB can be integrated in an HITI manner during the repair process.

Article Snippet: The Cas9-PBase fusion constructs were generated using hCas9_D10A (Addgene #41816) as the backbone, with the D10A nickase mutation reverted to wild-type D10 to restore full nuclease activity.

Techniques: Activity Assay