Decoupling deaminase recruitment and transgene targeting resolves tradeoffs in multiplexed base editing workflows

Engineering next-generation cell therapies can require multiple genetic modifications within the same workflow, including targeted transgene integration and multi-gene knockout, while preserving cell viability and genomic integrity. Conventional nuclease-based approaches can enable these modifications but rely on double-strand breaks, which can increase the risk of chromosomal translocations and imprecise donor integration.

In this ASGCT 2026 poster, Revvity demonstrates how the modular architecture of the Pin-point™ base editing platform can enable simultaneous targeted transgene integration and multiplexed base editing within a single engineering workflow.

The key strategy is to decouple deaminase recruitment from transgene targeting. By combining sgRNAs with and without aptamers, the nCas component can be directed to perform different functions within the same editing intervention:

• Transgene integration sites: aptamer-less sgRNAs generate paired nicks without recruiting the deaminase, enabling targeted transgene integration
• Base editing targets: aptamer-encoding sgRNAs recruit the deaminase to enable targeted base conversion and gene knockout

This mechanism is directly demonstrated on page 2: paired aptamer-less sgRNAs recruit nCas and generate two nicks while avoiding deaminase recruitment, allowing targeted integration from the donor template.

Key findings:

• High-efficiency multiplexed base editing achieved with minimal adverse effect on cell viability
• Precise targeted transgene integration achieved simultaneously with base editing by combining aptamer-encoding and aptamer-less sgRNAs
• Hypoimmunogenic iPSCs engineered with simultaneous transgene integration and base editing maintain genomic integrity
• Precise site-specific HLA-E transgene knock-in demonstrated at the B2M locus in clonal iPSCs
• Allogeneic CAR-T cells engineered in a single intervention by combining targeted CAR integration using AAV donors with base editing knockout of multiple genes
• Reduced translocations compared with SpCas9 nuclease editing
• Undetectable AAV donor integration at sgRNA-targeted sites with the Pin-point platform in the targeted hybridisation-capture analysis
• Edited cell products maintain key functional properties, including CAR-T cell killing activity and iPSC viability and pluripotency

The iPSC genomic-integrity and precise knock-in data are shown on page 4, alongside the single-step CAR-T engineering workflow. The poster also reports undetectable AAV donor integration at sgRNA-targeted sites with Pin-point in the targeted assay. And page 6 supports the cell-function statement: multiplexed Pin-point editing preserves iPSC live-cell count and Nanog expression, while the CAR-T cells retain killing activity.

Download the poster to explore the simultaneous transgene integration and multiplexed base editing workflow, genomic integrity data, and applications in engineered iPSCs and CAR-T cells.

The Pin-point™ base editing platform technology is available for clinical or diagnostic study and commercialization under a commercial license from Revvity.