FORGE-CRISPR: Reusable Modules for Focused CRISPR Library Construction

Abstract

Pooled CRISPR screening has become a widely used approach for functional genomics, yet the construction of screening libraries remains tightly coupled to specific vector architectures and experimental formats. Incorporation of barcodes, multiplexed guide configurations, alternative fluorophores, selectable markers, or distinct screening modalities often requires reconstruction of entire libraries, even when the underlying biological content remains unchanged. These limitations are particularly acute in engineered cellular systems that already contain reporters, knock-in alleles, or pre-existing selection markers. Here, we describe FORGE-CRISPR (Functional Oncology Research Genetic Engineering - CRISPR), a CRISPR library-construction system that separates biological guide content from screening-vector context. CRISPR knockout and CRISPR interference guide collections are first converted into reusable guide modules. Barcode modules and second-guide modules are generated separately and combined with guide modules during downstream assembly into screening acceptor vectors. This design allows guide-only, barcoded, and multiplexed libraries to be generated from shared physical components. To support this framework, we developed a manufacturing workflow in which synthetic oligonucleotide pools are converted into reusable FORGE module libraries through PCR amplification, Golden Gate cloning, and background suppression. Using this approach, we constructed nine reusable guide-module libraries ranging from 45 to 4,830 guides (11,303 guides in total), and evaluated library quality through PCR-NGS analysis of guide representation and abundance distributions. As content for these libraries, we defined a modular, non-overlapping set of focused target collections, termed the druggable oncology genome, by partitioning druggable targets according to clinical development status and dependency distribution in DepMap, while drawing guide sequences from established, experimentally validated genome-wide libraries. We report four resources: reusable guide, barcode, and second-guide modules; compatible screening acceptor vectors; a manufacturing and PCR-NGS quality-control workflow demonstrated across nine guide-module libraries; and a versioned set of focused druggable-oncology target collections.