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We thank all members of our laboratories for helpful comments and discussions related to this work. We also thank and acknowledge The Centre for Phenogenomics for all the help and expertise with the mouse work at the Lunenfeld-Tanenbaum Research Institute (LTRI), Toronto, Canada. This study was supported by a project grant from the Canadian Institutes of Health Research (PJT462506) and a Terry Fox Research Institute Program Projects Grant to D.S. (TFRI Project #1107) to D.S. and by the Nicol Family Foundation. E.R.L. was supported by the Canadian Cancer Society Research Training Award and the Frank Fletcher Memorial Fund.&#160;K.N.A. was supported by an H.L. Holmes Postdoctoral Award and Grants 1318698 and 26089 from the Cancer Research Society. J.N. was supported by a Canadian Institutes of Health Research Canada Graduate Scholarship &#8211; Master&#8217;s and a Canadian Institutes of Health Research Doctoral Research Award (#193389). Y.L was supported by the Canadian Institutes of Health Research&#8217;s Research Excellence, Diversity, and Independence fellowship (#ED6-190718).

D.S. is a consultant for and founder of ViVerita Therapeutics, and his lab has sponsored research agreements and service agreements with ViVerita Therapeutics.

A key consideration for pooled in vivo CRISPR screening is achieving sufficient library coverage while maintaining sparse, single-cell transduction to preserve clonal resolution2. In the mouse mammary gland, the epithelial compartment conservatively consists of approximately 3.5 &#215; 105 cells per gland36. At an average lentiviral transduction rate of 15%, which minimizes the frequency of multiple infections per cell, intraductal delivery results in ap...

Functional genomics screens provide a powerful framework to interrogate gene function at scale1. The development of CRISPR-Cas gene editing technologies has enabled the systematic perturbation of hundreds-to-thousands of genes in parallel, and pooled CRISPR-based screens in mammalian cell lines have become a cornerstone of modern cancer biology research2. These in vitro approaches have identified essential genes3, tumor suppressors4, and synthetic lethal interactions5 across diverse cancer contexts. However, one central limitation is an inability to capture the complex biological features of tumors in vivo, including interactions with the immune system and stroma, nutrient gradients, and tissue architecture within the microenvironment2. Consistent with this limitation, recent studies have demonstrated that many bona fide cancer driver genes fail to exert measurable effects in vitro, particularly when their function depends on microenvironmental cues that are absent in cell culture6,7,8,9,10.
The mouse mammary gland provides a uniquely advantageous tissue context for in vivo functional genomics screening. Mammary epithelial cells are highly regenerative, undergo rounds of clonal expansion during estrous cycling, and are organized into a branched ductal network that can be readily accessed and genetically manipulated11,12,13. These features have supported decades of mosaic genetic analyses and lineage-tracing experiments and establish the mammary gland as a tractable system for interrogating cell-intrinsic and microenvironment-dependent gene function during tissue homeostasis and disease14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33. Importantly, genetic perturbations can be restricted to the epithelial compartment, enabling direct study of epithelial-specific drivers of transformation, cell competition, lineage fidelity, and tumor progression in an intact tissue context.
While several strategies have been developed to deliver genetic perturbations directly to the adult mammary gland in situ, significant technical hurdles remain for high-throughput applications. Adenovirus-mediated delivery of Cre recombinase has been widely used to induce somatic recombination of conditional alleles in the adult mammary gland34,35,36,37, offering an efficient means to manipulate gene function without the complex germline breeding required for traditional genetically engineered mouse models (GEMMs). While high-titer adenovirus can also deliver CRISPR-Cas9 components to tissue-resident cells, these vectors do not integrate into the host genome38,39,40,41,42,43,44,45,46,47,48,49. This severely limits the scale of screens to ~10&#8211;55 genes, as each gRNA target site must be sequenced individually using molecular inversion probes38,39,40,41,42,43,44,45,46,47. Furthermore, current existing intraductal delivery methods enable direct access to the mammary ductal system but require either invasive surgical exposure of the mammary gland50 or rely on the use of comparatively&#160;large Hamilton syringes that are cumbersome51. While effective for localized delivery of virus, these approaches are comparatively invasive, technically challenging, and not optimized for scalable delivery of pooled lentiviral libraries.&#160;Collectively, these studies demonstrate that in situ viral perturbations are feasible, but current tools do not support the stable, pooled, sequence-retrievable perturbations required for high-throughput in vivo CRISPR screening.
To address these limitations, we and others have turned to integrating lentiviral vectors, which enable stable and heritable genetic modifications. In contrast to cell culture systems, in vivo tissues impose strict constraints on the number of perturbed cells that can be generated within a single organ, rendering genome-wide screening impractical. Consequently, in vivo CRISPR screens necessitate the design of biologically informed, targeted libraries that balance coverage with feasibility. Furthermore, most in vivo CRISPR screens, reviewed here2, have focused almost exclusively on lentiviral loss-of-function perturbations, despite increasing evidence that gene activation can reveal complementary and non-overlapping biological insights52,53,54,55. We and others have previously demonstrated that integrating lentiviral CRISPR perturbations into the adult mammary epithelium enables stable, heritable genetic modification in situ and that they can be recovered and quantified from genomic DNA36,55,56,57,58. Using this approach, we performed pooled in vivo CRISPR screens that identified new drivers of oncogenic transformation36,55, establishing both the feasibility and biological relevance of lentiviral delivery and CRISPR screening. However, these studies also highlighted the need for a simple, standardized, and scalable protocol that could be broadly adopted and extended to include both loss- and gain-of-function screening models within the same gland.
Here, we describe a simple and versatile intraductal injection strategy that enables parallel in vivo screening of CRISPR-knockout and activation libraries, termed CRISPR-KOALA, within the adult mouse mammary gland55. CRISPR-KOALA employs either a conventional 20-21 bp &#8216;single-guide&#8217; (sg)RNA for CRISPR-KO capabilities or a 14 bp &#8216;dead-guide&#8217; (dg)RNA for CRISPR-A. The dgRNA efficiently guides Cas9 to its target site but prevents Cas9 endonuclease activity59,60,61, and thus allows the use of a single mouse expressing active Cas9 for both knockout and activation modalities. Introducing two PP7 hairpins into the dgRNA trcr enables the recruitment of a PP7 Coat Protein:P65:HSF1 transactivator62 and leads to efficient overexpression of genes in vivo55 (Figure 1). By combining targeted gRNA library design with high-titer lentiviral production and non-surgical intraductal delivery using glass microcapillaries, this approach generates tens of thousands of spatially discrete epithelial clones while preserving tissue architecture and microenvironmental interactions. Importantly, lentiviral integration into the host genome enables the recovery and quantification of gRNAs via high-throughput sequencing, enabling pooled functional genomic screens in vivo. Together, this allows parallel or simultaneous (in the same cell) CRISPR-KO and CRISPR-A perturbations within the same mammary gland of a Cas9-expressing mouse.
We present a detailed step-by-step protocol encompassing gRNA library design, lentiviral production and concentration, intraductal delivery, library deconvolution, and data analysis (Figure 1). Together, this method provides a practical and scalable framework for in vivo CRISPR knockout and/or activation screening in the mouse mammary gland and can be adapted to study gene function during tissue homeostasis, different disease contexts, other tissues, and other species such as the rat.

<table><tbody><tr><td>0.45 micron filter</td><td>Sigma</td><td>S2HVU02RE</td><td></td></tr><tr><td>12k or 92k oligo chip</td><td>Customarray Inc. (Genscript)</td><td></td><td></td></tr><tr><td>15 cm cell culture plates</td><td>Corning</td><td>CLS353004</td><td></td></tr><tr><td>293FT</td><td>Invitrogen</td><td>R70007</td><td></td></tr><tr><td>293NT</td><td>Systems Biosciences</td><td>LV900A-1</td><td></td></tr><tr><td>Alkaline phosphatase</td><td>NEB</td><td>M0290L</td><td></td></tr><tr><td>Amplicillin</td><td>Fisher Scientific</td><td>BP1760-25</td><td></td></tr><tr><td>ATP</td><td>NEB</td><td>9804S</td><td></td></tr><tr><td>ATP</td><td>NEB</td><td>P0756S</td><td></td></tr><tr><td>Cutsmart buffer</td><td>NEB</td><td>B6004S</td><td></td></tr><tr><td>Deep sequencing (Next-Seq or Hi-Seq)</td><td>Illumina</td><td></td><td></td></tr><tr><td>DNAesy Blood and Tissue DNA extraction kit</td><td>Qiagen</td><td>69506</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium</td><td>Wisent</td><td>319-015-CL</td><td></td></tr><tr><td>Endura electrocompetent cells</td><td>Lucigen</td><td>60242-1</td><td></td></tr><tr><td>EpiCult-B Mouse Medium Kit</td><td>Stemcell Technologies</td><td>05610</td><td></td></tr><tr><td>Esp3I</td><td>NEB</td><td>R0734L</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Wisent</td><td>090-150</td><td></td></tr><tr><td>Gel DNA-cleanup kit</td><td>Zymo Research</td><td>D4008</td><td></td></tr><tr><td>Gene Pulser/MicroPulser Electroporation Cuvettes</td><td>BioRad</td><td>1652089</td><td></td></tr><tr><td>Gentle Collagenase/Hyaluronidase</td><td>Stemcell Technologies</td><td>7919</td><td></td></tr><tr><td>H11-Cas9</td><td>Jackson Labratories</td><td>JAX#028239</td><td></td></tr><tr><td>High-Speed Centrifuge</td><td>Beckman Coulter</td><td>MLS-50</td><td></td></tr><tr><td>Kim-wipe</td><td>Kimberly-Clark</td><td>34155</td><td></td></tr><tr><td>LB Agar</td><td>Wisent Technologies</td><td>800-011-LG</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>P97</td><td></td></tr><tr><td>Mini-prep plasmid Kit</td><td>Frogga Bio</td><td>PDH300</td><td></td></tr><tr><td>NEBuffer 3.1 (Buffer for BsmBI)</td><td>NEB</td><td>R0580L</td><td></td></tr><tr><td>Oligo Clean and Concentrator</td><td>Zymo Research</td><td>D4061</td><td></td></tr><tr><td>Oligo cleanup kit</td><td>Zymo research</td><td>D4060</td><td></td></tr><tr><td>PAGE purified illumina sequencing primer</td><td>IDT DNA</td><td></td><td></td></tr><tr><td>PCR Micropipettes</td><td>Drummond</td><td>5-000-1001-X10</td><td></td></tr><tr><td>PEI (polyethyleneimine)</td><td>Sigma</td><td>408727-100ML</td><td></td></tr><tr><td>Penicillin and Streptinomycin</td><td>Wisent</td><td>450-201-EL</td><td></td></tr><tr><td>pLKO-Cre</td><td>Addgene</td><td>158032</td><td></td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>12259</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>P2636-100MG</td><td></td></tr><tr><td>psPAX2</td><td>Addgene</td><td>12260</td><td></td></tr><tr><td>pXPR502-PPH-Cre</td><td>Addgene</td><td>256776</td><td></td></tr><tr><td>Q5 Polymerase 2x Master mix</td><td>NEB</td><td>M0494L</td><td></td></tr><tr><td>Qubit Fluorometric Quantification</td><td>Invitrogen</td><td>Q33327</td><td></td></tr><tr><td>R26-LSL-Cas9-EGFP</td><td>Jackson Labratories</td><td>JAX#024857</td><td></td></tr><tr><td>R26-LSL-TdTomato mice</td><td>Jackson Labratories</td><td>JAX#007909</td><td></td></tr><tr><td>SapI</td><td>NEB</td><td>R0569L</td><td></td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202L</td><td></td></tr><tr><td>Ultra-centrifuge tubes</td><td>Beckman Coulter</td><td>344058</td><td></td></tr><tr><td>Vacuum Filter Units</td><td>Fisher Scientific</td><td>SCHVU02RE</td><td></td></tr></tbody></table>

parallel in vivo screening of gene knockout and activation in the mouse mammary gland

Parallel In Vivo Screening of Gene Knockout and Activation in the Mouse Mammary Gland

Forward genetics screens are routinely employed to perturb thousands of genetic elements in a pooled fashion with the goal of producing large-scale genotype-to-phenotype maps. While often carried out in cell culture systems, accumulating evidence supports that in vivo screens have the power to unveil new biology that cannot be recapitulated in vitro. However, the widespread application of this approach has been limited by two major challenges: a predominant focus on loss-of-function perturbations rather than gene activation and the significant technical hurdles of delivering complex genetic libraries to specific tissues in vivo. To overcome these challenges, we describe a simple and versatile intraductal injection strategy that enables efficient and rapid functional genomic screening in the mouse mammary gland, by generating tens of thousands of discrete epithelial clones. Furthermore, we provide all the details necessary for library generation, intraductal injection, screen deconvolution, and analysis of CRISPR-Knockout and Activation libraries for comprehensive in vivo screens. Using these tools, which we termed CRISPR-KOALA (Knockout and Activation Linked Assay), we have identified new tumor suppressors and oncogenes within the coding and non-coding genome in pooled libraries ranging from 46 loci to one-fifth of the genome. Importantly, this approach and analysis can be applied to other organs to study the biological function of any gene during homeostasis or disease.

Here, we provide a protocol for pooled in vivo CRISPR screening in the mouse mammary gland. This pipeline can be used end-to-end to design and amplify pooled gRNA libraries from oligonucleotide arrays, clone the gRNA libraries into lentiviral backbones, produce high-titer lentivirus, perform intraductal delivery of pooled lentiviral libraries into the adult mammary gland, and recover gRNAs for sequencing-based deconvolution of library representation (Figure 1).
<p class="jove_co...

This protocol describes a scalable intraductal CRISPR screening approach enabling parallel in vivo gene knockout and activation studies in the mouse mammary gland.

Forward genetics screens are routinely employed to perturb thousands of genetic elements in a pooled fashion with the goal of producing large-scale ...

parallel-vivo-screening-gene-knockout-activation-mouse-mammary

Research

JoVE Journal

Genetics

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

Evaluation of Mammary Gland Development and Function in Mouse Models

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Here we describe the standard protocol for the detection of β-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

Intravital Subcellular Microscopy of the Mammary Gland

The present protocol describes intraductal injection of viral vectors via the teat to deliver genes of interest into the mammary epithelial cells.

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

Mount Sinai Hospital.  This protocol describes a scalable intraductal CRISPR screening approach enabling parallel in vivo gene knockout and activation studies in the mouse mammary gland.

Parallel In Vivo Screening of Gene Knockout and Activation in the Mouse Mammary Gland

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