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

We implemented CRISPR-StAR (Stochastic Activation by Recombination), an inducible pooled screening system that activates gene knockout after tumor engraftment and provides matched internal controls for guide-level normalization. Using barcode-embedded sequencing and Bayesian analysis, we screened a 30,000-sgRNA library in A549 xenografts and achieved reproducible dropout and enrichment phenotypes using just ~30 tumors.

Across additional xenograft models, single tumors yielded reliable functional annotation for ~1,000 genes leveraging within-tumor normalization. Comparing in vivo and in vitro screens uncovered tumor-suppressor effects detectable only in vivo; for example KMT2C and KMT2D knockouts produced contrasting tumor growth and transcriptional programs. Together with our R analysis package, CRISPR-StAR enables scalable in vivo dependency mapping that complements in vitro resources and reduces animal use up to sevenfold versus conventional dropout screens, improving overall methodological rigor at genome-scale clonal resolution.