LandingPad™ Gene Integration Platform
Precise, large-fragment, and iterative integration — engineered cell models built for research.
LandingPad™: Turn Cell-Line Construction into a Reusable Engineering Platform
• Build an attP landing-pad cell line once, then integrate additional genes in a streamlined step without redesigning sgRNAs or repeating monoclonal screening for every payload.
• Support site-specific integration of DNA fragments larger than 10 kb, including large genes and multi-gene circuits that can be difficult to accommodate with conventional methods.
• Use a genomic attP site, an attB donor, and Bxb1 recombinase for directional integration. The reaction is stable and does not normally reverse without an additional directionality factor (RDF).
• Applications include isogenic stable cell lines, large-gene disease models, reporter systems, iPSCs, and engineered cell-therapy models.
Redefining Gene Integration: Build the Platform Once, Load It Repeatedly
Stable expression models underpin functional genomics, disease-mechanism research, drug-target validation, and synthetic biology. Researchers often need more than a single clone: they need reusable models with a consistent genetic background and comparable expression.
Conventional approaches face several limitations. Random integration can cause position effects and large clone-to-clone expression differences. HDR-mediated knock-in efficiency often declines as insert size increases, making large genes such as DMD, RYR1, and ATM difficult to introduce. Each new gene may require another round of guide design and clone screening, while multi-gene pathways are difficult to rebuild with controlled component ratios.
LandingPad™ addresses these challenges with a two-stage workflow. First, Flash-KI or the Bingo™ prime-editing platform is used to install an attP landing sequence at a safe-harbor locus such as AAVS1, or at a specified disease- or therapy-relevant locus. Following clonal screening and validation, the resulting landing-pad cells can be reused. Second, an attB-containing donor and Bxb1 recombinase are introduced to integrate a new payload at the predefined site.
Bxb1 is a large serine recombinase. After recombination, the resulting attL/attR sites are not substrates for the enzyme under standard conditions, so reverse excision does not normally occur unless a directionality factor is supplied. LandingPad™ is therefore designed as a reusable cellular foundation rather than a one-time integration tool.
EDITGENE's Platform-Level Capabilities
Large-fragment, site-specific integration
Isogenic model development
Reusable landing-pad cells
Broad cell-type coverage
Integrated workflow
Experienced Team
Applications
With conventional stable cell lines, random integration can cause expression to vary several-fold between clones, and expression may drift or become silenced during long-term culture. These differences make it difficult to determine whether a phenotype reflects the tested mutation or variation in integration site and copy number. LandingPad™ places each payload at the same prevalidated locus to reduce this source of variation.
Research challenges: Large clone-to-clone expression differences, unreliable comparisons between variants in different genetic backgrounds, and expression drift during passaging.
• Landing-pad installation: Flash-KI or Bingo™ prime editing introduces attP at loci such as AAVS1. Clones are screened and validated by PCR and Sanger sequencing, then delivered as cryopreservable, reusable cell stocks.
• Donor engineering: promoters, linkers, and selection markers are configured to meet expression goals, with single-copy or multi-copy designs and weaker promoters available to avoid excessive expression.
• Integration and stability testing: attB donors are integrated at the same locus, integration is quantified by high-throughput sequencing, and expression stability is evaluated across serial passages. Cell lines can be supplied without continuous drug selection.
• Isogenic variant panels: wild-type and mutant variants—including kinase-dead, domain-deletion, and point-mutant series—integrated at the same locus.
• Parallel expression of paralogs: compare functional redundancy and specificity among related genes in a consistent background.
• Reporter and inducible-expression cell lines: combine target proteins with fluorescent, luminescent, or selection reporters, optionally using Tet-On/Tet-Off regulation for screening and dose-response studies.
HDR-mediated knock-in efficiency often declines sharply for inserts above a few kilobases, while viral vectors have packaging limits. As a result, reliable cDNA replacement or complementation models remain difficult for some large disease genes. Bxb1-mediated LandingPad™ integration provides a route for targeted insertion of DNA fragments larger than 10 kb.
| Payload Size | Representative Genes | Conventional Challenge | LandingPad™ Approach |
|---|---|---|---|
| >10 kb | DMD (~11 kb), BRCA2 (~10 kb) | Very low HDR efficiency; viral packaging constraints | Bxb1-mediated large-payload integration using suitable donor formats |
| 5–10 kb | ATM (~9 kb), FBN1 (~8.6 kb), CACNA1A (~7.5 kb), ABCA4 (~6.8 kb), SCN1A (~6 kb) | Declining HDR efficiency and extensive clone screening | Targeted integration at a validated landing site |
| 1–5 kb | CFTR (~4.4 kb), FANCA (~4.4 kb), GBA1 (~1.6 kb), F9 (~1.4 kb), SMN1 (~0.9 kb) | Repeated construction across genetic backgrounds | Reuse the same attP landing-pad cells to shorten later projects |
Research needs: Large-gene complementation models for genes such as DMD and RYR1; systematic comparison of pathogenic variants across protein domains; reconstruction of multi-gene pathways; and more practical large-gene models in iPSCs.
• Large-payload integration: support fragments over 10 kb, with donor preparation and quality control designed to help preserve payload integrity.
• Sequential integration: integrate two to four components sequentially at the same landing-pad locus when appropriate for pathway reconstruction or co-expression of complex subunits.
• Delivery and validation: select plasmid, IDLV, or other donor formats according to the cell type; combine FLASH delivery with expression testing by Western blot, flow cytometry, or qPCR and functional complementation assessment.
• Large-gene knock-in lines pairing wild-type and patient-derived variants, with deletion or truncation series for complementation and structure–function analysis.
• Multi-gene pathway models that reconstruct signaling or DNA-repair pathways in the same cell.
• iPSC disease models in which attP is installed before differentiation and the disease-associated gene is integrated later, helping address clonal-screening bottlenecks.
Synthetic biology and pathway studies often require multiple transcription units to be expressed at defined copy numbers and ratios, with stable expression over time. A published multi-landing-pad platform in CHO cells demonstrated Bxb1-mediated integration of up to approximately 100 kb of exogenous DNA containing 21 transcription units, with stable recombinant-protein expression over several weeks and antibody expression increasing with integration copy number. LandingPad™ applies the same general principle to reusable, controllable cellular platforms.
Common challenges: Uncontrolled copy number after random integration, imbalanced expression among transcription units, repeated cell-line reconstruction for each circuit version, and reporter-signal drift during long-term culture.
• Multi-site landing-pad cells: install multiple attP sites when needed to support parallel circuits, with each site evaluated for stable expression.
• Modular donor design: configure promoters, insulators, terminators, and selection markers, with controlled single- or multi-copy designs.
• Long-term stability testing: assess expression across serial passages. Circuit versions can be changed by replacing the attB donor rather than rebuilding the host cell.
• Pathway reporter lines for quantitative pathway activity measurements and high-throughput screening.
• Multi-gene circuit lines that allow rapid iteration of transcription factors, effectors, and regulatory modules on the same cellular background.
• Recombinant protein or antibody expression lines with controlled multi-copy integration and stable expression, potentially serving as copy-number-defined reference models.
A key goal in cell-therapy engineering is to place therapeutic payloads at defined genomic sites with controlled expression rather than relying solely on random viral integration. For example, targeted insertion of a CAR cassette at the TRAC locus can place CAR expression under the control of the endogenous TCR promoter. By contrast, B2M is commonly disrupted to reduce immune recognition in allogeneic cells; it serves a different purpose and should not be confused with a typical payload-integration site.
Research challenges: Variable CAR copy number and expression after random integration, repeated testing of combinations such as CAR plus cytokine armor and safety switches, and the need to balance editing efficiency with viability in primary T cells and iPSCs.
• Locus assessment and landing-pad installation: evaluate TRAC, AAVS1, or other suitable loci for the cell type, install attP, and then integrate CAR or other payloads through Bxb1.
• Primary-cell delivery: optimize electroporation or viral delivery parameters using the FLASH platform, balancing efficiency and viability; IDLV-format donors may be considered.
• Integration and functional testing: quantify integration and CAR expression by NGS and flow cytometry, with in vitro cytotoxicity testing where appropriate. Directional integration helps prevent previously integrated payloads from being excised by subsequent standard reactions.
• T-cell or iPSC landing-pad models with attP at TRAC for site-specific integration of CD19-CAR, BCMA-CAR, and related payloads.
• Multi-payload cell lines combining CARs, cytokine armor, and safety switches for iterative optimization.
• iPSC-derived NK/T-cell precursor models for studying endogenous-promoter expression dynamics and exhaustion-related phenotypes in preclinical concept validation.
Technical Principle: Why Bxb1 and Why Install attP?
Bxb1 Origin and Performance
The Bxb1 catalytic component used by EDITGENE is an engineered variant derived through phage-assisted continuous evolution (PACE) of wild-type Bxb1 large serine recombinase, followed by rational combination of beneficial mutations across domains. Published work reports higher activity than wild-type Bxb1.
• In cells with preinstalled landing sites, integration of a 5.6 kb donor reached up to approximately 60% in a reported setting, about 3.2-fold higher than wild-type Bxb1.
• In a single-transfection setup, reported average targeted integration was approximately 23%, around 4.2-fold higher than wild type; efficiencies above 30% were reported at multiple sites in primary human fibroblasts.
• Compared with PASTE, a prime-editing/recombinase-fusion strategy, the reported average was approximately 16-fold higher.
These figures come from published experiments under specific targets, cell lines, and delivery conditions. Actual efficiency varies with locus, cell type, delivery method, and donor design. EDITGENE evaluates feasibility and may conduct project-specific pilot studies; no unconditional efficiency is implied.
A Key Design Rule: Install attP in the Genome, Not attB
The recommended configuration is genomic attP + an attB-containing donor + Bxb1 recombinase. Reported off-target analyses found that installing attB in the genome could lead Bxb1 variants to integrate at highly homologous genomic sequences, whereas genomic attP was associated with very low off-target integration.
Combined with Bxb1's directional recombination behavior, the resulting attL/attR sites are not normally excised without a directionality factor. This supports stable integration without the need for continuous drug selection.
LandingPad™ Compared with Flp-In™ T-REx™ and Cre-loxP
| Comparison | Flp-In™ T-REx™ | Cre-loxP | LandingPad™ (Bxb1) |
|---|---|---|---|
| Recombination system | Flp / FRT | Cre / loxP | Bxb1 / attB and attP |
| Directionality and stability | Potentially reversible; continued selection may be needed | Can be reversible; same-direction loxP sites may be excised by Cre | Directional integration; attL/attR are not normally reversed without RDF |
| Host-cell flexibility | Often relies on commercial Flp-In cells | Can be built in-house, with Cre expression requiring control | attP landing-pad cells can be developed for different cell types |
| Large-payload capacity | Generally limited | Generally limited | Supports fragments over 10 kb |
| Off-target considerations | Generally low in intended systems | Cre may recognize genomic pseudo-loxP sites | Very low reported off-target integration when genomic attP is used |
| Typical applications | Stable cell lines | Conditional knockout and recombination tools | Isogenic models, large-gene disease models, gene circuits, and CAR-T engineering |
EDITGENE Platform Advantages
The value of LandingPad™ comes not only from the recombinase, but also from integrating landing-site installation, donor engineering, delivery, and clone screening into a coordinated workflow.
Flash-KI targeted integration platform
Installs attP precisely at safe-harbor or specified endogenous loci and supports large donors and multiple cell types.
Bingo™ prime-editing platform
Offers an alternative route for point mutations or selected installations without relying on double-strand breaks, potentially useful for DNA-damage-sensitive cells such as primary cells and iPSCs.
Bxb1 large-payload system
Uses an engineered recombinase variant and the genomic attP + donor attB configuration to support integration of fragments over 10 kb while managing off-target risk.
FLASH delivery platform
Adapts plasmid, RNP, IDLV, and other delivery formats to challenging-to-transfect cells, primary cells, and stem cells.
Monoclonal screening platform
Screens, expands, and characterizes landing-pad and payload-integrated clones, with clear genetic-background and purity information.
Project experience
More than 3,000 gene-editing CRO projects across 400+ cell types, supporting end-to-end project delivery.
LandingPad™ Service Workflow and Deliverables
• Assess target locus and design the strategy, including safe-harbor loci such as AAVS1 or therapy-related/specified endogenous loci such as TRAC.
• Install attP using Flash-KI or Bingo™ prime editing; confirm by PCR screening and Sanger sequencing; deliver cryopreservable, reusable landing-pad cells with a validation report.
• Design and construct attB donors in vector or large-fragment formats; provide Bxb1 recombinase as an expression vector, protein, or mRNA where appropriate, with integration guidance and optimization.
• Quantify integration by high-throughput sequencing; monoclonal screening and expansion can be added, including verification of the desired genotype.
• Deliverables may include the attP landing-pad cell line, attB donor vector or large-fragment donor, Bxb1 expression construct, integration-efficiency report, monoclonal sequencing validation report when clone screening is selected, and an operating SOP.
• Building the attP landing-pad cell line typically takes 8–12 weeks. Subsequent payload integration and verification may take approximately 1–2 weeks; monoclonal screening and expansion require additional assessment.
Frequently Asked Questions
Q1. How long does it take to integrate a gene after the attP landing-pad cell line is established?
Q2. Can the attP landing-pad cells be reused?
Q3. Why install attP in the genome rather than attB?
Q4. What integration efficiencies can Bxb1 achieve?
Q5. Which cell types can be used to build attP landing-pad lines?
Q6. What are the main differences between LandingPad™, Flp-In™, and Cre-loxP?
Make LandingPad™ Your Reusable Cellular Foundation
Whether you need an isogenic variant panel, a large-gene disease model, an iteratively updated gene circuit, or a reusable CAR-engineering platform, EDITGENE can support locus design, donor engineering, delivery optimization, clone validation, and functional assessment. Let an experienced engineering platform manage the technical details of cell-line construction so your team can focus on the scientific questions.
