FORGE-KI: A Modular Framework for Endogenous Knock-In Engineering Across HDR and PITCh/MMEJ Repair Pathways

Targeted knock-in technologies have enabled precise insertion of reporters, affinity tags, degrons, and other functional payloads into endogenous genomic loci. Over the past decade, a diverse collection of genome engineering strategies has emerged, including approaches based on homology-directed repair (HDR), microhomology-mediated end joining (MMEJ), homology-mediated end joining (HMEJ), and related methodologies. While these advances have greatly expanded the capabilities of endogenous genome engineering, they have also increased the complexity of donor design, assembly, and validation.

Here, we describe FORGE-KI (Functional Oncology Research Genetic Engineering – Knock in), a pathway-matched design workflow for endogenous knock-in engineering that aligns the assembly strategy with the underlying repair mechanism. For large-cargo insertions, we use a modular five-component framework that separates gene-specific targeting arms from reusable functional modules, allowing rapid assembly of HDR donor constructs targeting AHR, IRF1, and FOSL1 from a shared reagent collection. For MMEJ/PITCh applications, where short targeting elements permit rapid fabrication, we developed a streamlined one-step pipeline in which the entire donor and selection payload is synthesized as a single continuous fragment for direct cloning, compressing the design-to-reagent cycle time. This MMEJ workflow is paired with a dual-promoter nuclease vector (pForge-KI-MMEJ-Cas9-DualGuide) that drives the PITCh-release and locus-specific guides from distinct promoters, a design intended to reduce the repeated-promoter instability associated with some dual-guide vectors.

We also established a standardized workflow for donor assembly, generation of knock-in cell populations, molecular validation, and selectable-cassette removal, and we demonstrate it by generating a functional, selection-marker-free, cytokine-inducible IRF1 HDR reporter line and an inducible IRF1 PITCh/MMEJ reporter pool with confirmed junction enrichment. In parallel, we developed forgeKI, an R package that automates C-terminal reporter knock-in design across both HDR and PITCh/MMEJ repair pathways, including guide selection, target-biology validation, targeting-arm design, domestication, donor-assembly planning, and generation of synthesis-ready constructs.

Together, the reagents and software provide a practical system for endogenous knock-in engineering that supports multiple payloads, selection strategies, and repair pathways within a shared donor organization. Rather than replacing existing knock-in technologies, this framework provides a modular foundation for incorporating, extending, and automating the published knock-in methods.