Temporal control of sgRNA library activation unlocks large-scale in vivo CRISPR screens

Functional genomics screens have illuminated genetic dependencies in cancer, but conventional in vitro approaches fail to capture vulnerabilities shaped by the tumor microenvironment. Here, we implement CRISPR-StAR (Stochastic Activation by Recombination), a next-generation inducible CRISPR screening platform for large-scale in vivo applications. The system uses a dual lox-based recombination system to enable guide-level normalization and clonal knockout phenotyping. To analyze the rich (barcode-embedded) sequencing output, we developed UMIBB, a superior Bayesian statistical framework for quantifying gene-level dropout and enrichment compared to conventional software packages. Screening a 30,000-sgRNA library in A549 xenografts, followed by clone representation and dropout correlation analyses, showed high fidelity and reproducibility with dropout phenotypes resolvable using as few as 30 tumors for this size library. Validation across multiple tumor models demonstrated that a single tumor can provide reliable, functional annotation for [~]1,000 genes leveraging intra-tumor library controls for normalization. Comparing in vivo and in vitro screens revealed that a substantial subset of tumor suppressor genes exerts strong phenotypic effects only observable in vivo. For example, single-gene knockout and transcriptomic profiling confirmed that KMT2C and KMT2D have contrasting impacts on tumor growth - an insight that would have been overlooked in standard cell culture. Looking ahead, CRISPR-StAR screening, combined with our user-friendly analysis pipeline available on GitHub (R-package), offer an integrated framework for creating in vivo dependency maps that can complement existing vitro datasets like DepMap and Achilles. Critically, our approach reduces animal use by up to 7-fold compared to conventional in vivo dropout screens. This represents a significant ethical and methodological advancement - achieving genome-scale resolution with far fewer animals and greater reproducibility.