CRISPR-StAR (stochastic activation by recombination) is an inducible pooled screening system that activates gene knockout after tumor engraftment and provides matched internal controls for guide-level normalization. In this study, we explore the scalability and reproducibility of this approach for in vivo cancer screens. Through barcode-embedded sequencing and the development of a Bayesian analysis pipeline, we screened a 30,000-sgRNA library in A549 xenografts, achieving reproducible dropout and enrichment phenotypes using just ∼30 tumors. Across additional xenograft models, single tumors yielded reliable functional annotation for ∼1,000 genes. Comparing in vivo and in vitro screens uncovered tumor suppressor effects detectable only in vivo; for example, KMT2C and KMT2D knockouts produced contrasting growth and transcriptional programs. Together with our R analysis package, we show that CRISPR-StAR enables scalable in vivo dependency mapping that complements in vitro resources and reduces animal use by up to 7-fold versus conventional dropout screens, improving methodological rigor at genome-scale clonal resolution.

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. ### Competing Interest Statement All authors were consultants, shareholders and/or employees of Tango Therapeutics at the time of their contributions to this body of work. U.E., D.S., and X.P. are also co-founders and shareholders of ViVerita Therapeutics. Tango Therapeutics (United States), https://ror.org/003x6t567

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that induce selective protein degradation by linking an E3 ubiquitin ligase enzyme to a target protein. This approach allows scope for targeting “undruggable” proteins, and several PROTACs have reached the stage of clinical candidates. However, the roles of cellular transmembrane transporters in PROTAC uptake and efflux remain underexplored. Here, we utilized transporter-focused genetic screens to identify the ATP-binding cassette transporter ABCC1/MRP1 as a key PROTAC resistance factor. Unlike the previously identified inducible PROTAC exporter ABCB1/MDR1, ABCC1 is highly expressed among cancers of various origins and constitutively restricts PROTAC bioavailability. Moreover, in a genome-wide PROTAC resistance screen, we identified candidates involved in processes such as ubiquitination, mTOR signaling, and apoptosis as genetic factors involved in PROTAC resistance. In summary, our findings reveal ABCC1 as a crucial constitutively active efflux pump limiting PROTAC efficacy in various cancer cells, offering insights for overcoming drug resistance.

Pooled genetic screening with CRISPR-Cas9 has enabled genome-wide, high-resolution mapping of genes to phenotypes, but assessing the effect of a given genetic perturbation requires evaluation of each single guide RNA (sgRNA) in hundreds of cells to counter stochastic genetic drift and obtain robust results. However, resolution is limited in complex, heterogeneous models, such as organoids or tumors transplanted into mice, because achieving sufficient representation requires impractical scaling. This is due to bottleneck effects and biological heterogeneity of cell populations. Here we introduce CRISPR-StAR, a screening method that uses internal controls generated by activating sgRNAs in only half the progeny of each cell subsequent to re-expansion of the cell clone. Our method overcomes both intrinsic and extrinsic heterogeneity as well as genetic drift in bottlenecks by generating clonal, single-cell-derived intrinsic controls. We use CRISPR-StAR to identify in-vivo-specific genetic dependencies in a genome-wide screen in mouse melanoma. Benchmarking against conventional screening demonstrates the improved data quality provided by this technology.

CRISPR-mediated genome editing is a powerful approach to understanding disease biology, including identifying genes essential for cancer cell proliferation, immune evasion and survival. We routinely use the technology to identify novel therapeutic targets for cancer using both in vitro and in vivo tumor models, focusing on targets that are synthetic lethal with tumor suppressor gene loss. Large-scale CRISPR screens are often conducted using in vitro cell culture systems as in vivo ‘drop-out’ screens face several bottlenecks resulting in poor sgRNA library representation and low signal to noise ratio. These inherent limitations of in vivo screens occur because only a small fraction of the injected cells contribute to xenograft tumor formation, and because of the uneven clonal expansion due to heterogenous growth conditions in the tumor microenvironment. Hence, many in vivo screens are underpowered for statistical analysis, resulting in a high rate of ‘false negatives’ or ‘false positives’ unless a large number of animals are used or the size of the sgRNA library is greatly reduced to overcome sampling noise. Here, we validate a novel in vivo screening technology, CRISPR-StAR (Stochastic Activation by Recombination), that overcomes these challenges by (i) activating the sgRNA library in established tumors and (ii) generating internally matched-pair controls for each sgRNA using molecular barcodes to capture the history of each clone within the tumor. Using this clonal information, we developed a robust computational pipeline that extracts meaningful target sgRNA-level data from individual tumors, despite the random under-representation of the larger library. Statistical (down-sampling) analysis revealed that CRISPR-StAR has a resolution of 1,000 sgRNAs per tumor which reduces the number of animals required by 7-fold to traditional approaches. Consequently, we have performed several druggable genome screens (~30,000 sgRNA) using just 30-40 individual tumors and identified a catalog of tumor suppressor genes that, when lost, strongly promote tumor growth in vivo without affecting cell proliferation in vitro. This group of genes is enriched with epigenetic modifiers, in particular multiple members of the COMPASS family and the SWI/SNF chromatin remodeling complexes. These results validate CRISPR-StAR as a powerful in vivo functional genomics platform for high throughput target discovery screens. Citation Format: Silvia Fenoglio, James Tepper, Lauren Grove, Esther CH Uijttewaal, Alborz Bejnood, Yi Yu, Hsin-Jung Wu, Annabel Devault, Shangtao Liu, Binzhang Shen, Samuel R Meier, Ashley H Choi, Tenzing Khendu, Hannah Stowe, Minjie Zhang, Brian B Haines, Alan Huang, Jannik N Andersen, Xuewen Pan, Ulrich Elling, Teng Teng. Inducible activation of sgRNA libraries in tumor xenografts empowers large-scale in vivo target discovery screens [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C048.

Genomic imprinting is regulated by parental-specific DNA methylation of imprinting control regions (ICRs). Despite an identical DNA sequence, ICRs can exist in two distinct epigenetic states that are memorized throughout unlimited cell divisions and reset during germline formation. Here, we systematically study the genetic and epigenetic determinants of this epigenetic bistability. By iterative integration of ICRs and related DNA sequences to an ectopic location in the mouse genome, we first identify the DNA sequence features required for maintenance of epigenetic states in embryonic stem cells. The autonomous regulatory properties of ICRs further enabled us to create DNA-methylation-sensitive reporters and to screen for key components involved in regulating their epigenetic memory. Besides DNMT1, UHRF1 and ZFP57, we identify factors that prevent switching from methylated to unmethylated states and show that two of these candidates, ATF7IP and ZMYM2, are important for the stability of DNA and H3K9 methylation at ICRs in embryonic stem cells.

Millions of putative transcriptional regulatory elements (TREs) have been cataloged in the human genome, yet their functional relevance in specific pathophysiological settings remains to be determined. This is critical to understand how oncogenic transcription factors (TFs) engage specific TREs to impose transcriptional programs underlying malignant phenotypes. Here, we combine cutting edge CRISPR screens and epigenomic profiling to functionally survey ≈15,000 TREs engaged by estrogen receptor (ER). We show that ER exerts its oncogenic role in breast cancer by engaging TREs enriched in GATA3, TFAP2C, and H3K27Ac signal. These TREs control critical downstream TFs, among which TFAP2C plays an essential role in ER-driven cell proliferation. Together, our work reveals novel insights into a critical oncogenic transcription program and provides a framework to map regulatory networks, enabling to dissect the function of the noncoding genome of cancer cells.

Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12‐dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT‐AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT‐AN minor introns in ˜ 100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing and suggest a link between minor spliceosome defects and constitutional aneuploidy in humans. Genetic causes of aneuploidy in humans remain largely unknown. Here, patient exome sequencing reveals pathogenic patient mutations in CENATAC/CCDC84, encoding a novel component of the minor spliceosome, and downstream effects on chromosome segregation in mitosis. Biallelic CCDC84/CENATAC mutations identified through patient exome sequencing link altered minor intron splicing to constitutional mosaic aneuploidy in humans.

To understand how chromatin domains coordinate gene expression, we dissected select genetic elements organizing topology and transcription around the Prdm14 super enhancer in mouse embryonic stem cells. Taking advantage of allelic polymorphisms, we developed methods to sensitively analyze changes in chromatin topology, gene expression, and protein recruitment. We show that enhancer insulation does not rely strictly on loop formation between its flanking boundaries, that the enhancer activates the Slco5a1 gene beyond its prominent domain boundary, and that it recruits cohesin for loop extrusion. Upon boundary inversion, we find that oppositely oriented CTCF terminates extrusion trajectories but does not stall cohesin, while deleted or mutated CTCF sites allow cohesin to extend its trajectory. Enhancer-mediated gene activation occurs independent of paused loop extrusion near the gene promoter. We expand upon the loop extrusion model to propose that cohesin loading and extrusion trajectories originating at an enhancer contribute to gene activation.

Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC ( CCDC84 ). We show that CENATAC is a novel component of the minor (U12-dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT-AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT-AN minor introns in ~100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing with a specific impact on the chromosome segregation process, and show how defects herein can cause constitutional aneuploidy.### Competing Interest StatementNazneen Rahman is a Non-Executive Director of AstraZeneca. The other authors declare no competing interests.

CRISPR-Cas9 is an efficient and versatile tool for genome engineering in many species. However, inducible CRISPR-Cas9 editing systems that regulate Cas9 activity or sgRNA expression often suffer from significant limitations, including reduced editing capacity, off-target effects, or leaky expression. Here, we develop a precisely controlled sgRNA expression cassette that can be combined with widely-used Cre systems, termed CRISPR-Switch (SgRNA With Induction/Termination by Cre Homologous recombination). Switch-ON facilitates controlled, rapid induction of sgRNA activity. In turn, Switch-OFF-mediated termination of editing improves generation of heterozygous genotypes and can limit off-target effects. Furthermore, we design sequential CRISPR-Switch-based editing of two loci in a strictly programmable manner and determined the order of mutagenic events that leads to development of glioblastoma in mice. Thus, CRISPR-Switch substantially increases the versatility of gene editing through precise and rapid switching ON or OFF sgRNA activity, as well as switching OVER to secondary sgRNAs.