Abstract
Here we provide a detailed tutorial on CRISPR in vivo screening. Using the mouse as the model organism, we introduce a range of CRISPR tools and applications, delineate general considerations for ‘transplantation-based’ or ‘direct in vivo’ screening design, and provide details on technical execution, sequencing readouts, computational analyses and data interpretation. In vivo screens face unique pitfalls and limitations, such as delivery issues or library bottlenecking, which must be counteracted to avoid screening failure or flawed conclusions. A broad variety of in vivo phenotypes can be interrogated such as organ development, hematopoietic lineage decision and evolutionary licensing in oncogenesis. We describe experimental strategies to address various biological questions and provide an outlook on emerging CRISPR applications, such as genetic interaction screening. These technological advances create potent new opportunities to dissect the molecular underpinnings of complex organismal phenotypes.
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Data availability
The main data discussed in this protocol are available in the supporting primary research papers (https://doi.org/10.1016/j.cell.2015.02.038 and https://doi.org/10.1073/pnas.1600582113).
References
Weber, J., Braun, C. J., Saur, D. & Rad, R. In vivo functional screening for systems-level integrative cancer genomics. Nat. Rev. Cancer 20, 573–593 (2020).
Gilbert, L. A. & Hemann, M. T. DNA damage-mediated induction of a chemoresistant niche. Cell 143, 355–366 (2010).
Bent, E. H., Gilbert, L. A. & Hemann, M. T. A senescence secretory switch mediated by PI3K/AKT/mTOR activation controls chemoprotective endothelial secretory responses. Genes Dev. 30, 1811–1821 (2016).
Schneider, G., Schmidt-Supprian, M., Rad, R. & Saur, D. Tissue-specific tumorigenesis: context matters. Nat. Rev. Cancer 17, 239–253 (2017).
Beronja, S. et al. RNAi screens in mice identify physiological regulators of oncogenic growth. Nature 501, 185–190 (2013).
LaFleur, M. W. et al. A CRISPR–Cas9 delivery system for in vivo screening of genes in the immune system. Nat. Commun. 10, 1668 (2019).
Wuestefeld, T. et al. A direct in vivo RNAi screen identifies MKK4 as a key regulator of liver regeneration. Cell 153, 389–401 (2013).
Rogers, Z. N. et al. A quantitative and multiplexed approach to uncover the fitness landscape of tumor suppression in vivo. Nat. Methods 14, 737–742 (2017).
Braun, C. J. et al. Versatile in vivo regulation of tumor phenotypes by dCas9-mediated transcriptional perturbation. Proc. Natl. Acad. Sci. USA. 113, E3892–E3900 (2016).
Chen, S. et al. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160, 1246–1260 (2015).
Evers, B. et al. CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes. Nat. Biotechnol. 34, 631–633 (2016).
Sanson, K. R. et al. Optimized libraries for CRISPR–Cas9 genetic screens with multiple modalities. Nat. Commun. 9, 1–15 (2018).
Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).
Wang, T. et al. Identification and characterization of essential genes in the human genome. Science 350, 1096–1101 (2015).
Aguirre, A. J. et al. Genomic copy number dictates a gene-independent cell response to CRISPR/Cas9 targeting. Cancer Discov. 6, 914–929 (2016).
Munoz, D. M. et al. CRISPR screens provide a comprehensive assessment of cancer vulnerabilities but generate false-positive hits for highly amplified genomic regions. Cancer Discov. 6, 900–913 (2016).
Meyers, R. M. et al. Computational correction of copy number effect improves specificity of CRISPR–Cas9 essentiality screens in cancer cells. Nat. Genet. 49, 1779–1784 (2017).
Liu, S. J. et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355, eaah7111 (2017).
Zhou, H. et al. In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice. Nat. Neurosci. 21, 440–446 (2018).
Roth, T. L. et al. Pooled knockin targeting for genome engineering of cellular immunotherapies. Cell 181, 728–744.e21 (2020).
Nakamura, M., Gao, Y., Dominguez, A. A. & Qi, L. S. CRISPR technologies for precise epigenome editing. Nat. Cell Biol. 23, 11–22 (2021).
Nissim, L., Perli, S. D., Fridkin, A., Perez-Pinera, P. & Lu, T. K. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol. Cell 54, 698–710 (2014).
McCarty, N. S., Graham, A. E., Studená, L. & Ledesma-Amaro, R. Multiplexed CRISPR technologies for gene editing and transcriptional regulation. Nat. Commun. 11, 1281 (2020).
Labuhn, M. et al. Refined sgRNA efficacy prediction improves large- and small-scale CRISPR–Cas9 applications. Nucleic Acids Res. 46, 1375–1385 (2018).
Wessels, H.-H. et al. Massively parallel Cas13 screens reveal principles for guide RNA design. Nat. Biotechnol. 38, 722–727 (2020).
Horlbeck, M. A. et al. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife 5, e19760 (2016).
Hanna, R. E. & Doench, J. G. Design and analysis of CRISPR–Cas experiments. Nat. Biotechnol. 38, 813–823 (2020).
Montague, T. G., Cruz, J. M., Gagnon, J. A., Church, G. M. & Valen, E. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42, 401–407 (2014).
Heigwer, F., Kerr, G. & Boutros, M. E-CRISP: fast CRISPR target site identification. Nat. Methods 11, 122–123 (2014).
Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR–Cas9. Nat. Biotechnol. 34, 184–191 (2016).
Concordet, J. P. & Haeussler, M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 46, W242–W245 (2018).
Meier, J. A., Zhang, F. & Sanjana, N. E. GUIDES: sgRNA design for loss-of-function screens. Nat. Methods 14, 831–832 (2017).
Li, F. et al. In vivo epigenetic crispr screen identifies asf1a as an immunotherapeutic target in kras-mutant lung adenocarcinoma. Cancer Discov. 10, 270–287 (2020).
Wu, Q. et al. In vivo CRISPR screening unveils histone demethylase UTX as an important epigenetic regulator in lung tumorigenesis. Proc. Natl. Acad. Sci. USA. 115, E3978–E3986 (2018).
Michels, B. E. et al. Pooled in vitro and in vivo CRISPR–Cas9 screening identifies tumor suppressors in human colon organoids. Cell Stem Cell 26, 782–792.e7 (2020).
Becker, M. et al. CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries. Nucleic Acids Res. 48, e78 (2020).
Charan, J. & Kantharia, N. D. How to calculate sample size in animal studies? J. Pharmacol. Pharmacother. 4, 303 (2013).
Manguso, R. T. et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature 547, 413–418 (2017).
Ye, L. et al. In vivo CRISPR screening in CD8 T cells with AAV–Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma. Nat. Biotechnol. 37, 1302–1313 (2019).
Zender, L. et al. An oncogenomics-based in vivo RNAi screen identifies tumor suppressors in liver cancer. Cell 135, 852–864 (2008).
Yuen, G. et al. CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level. Nucleic Acids Res. 45, 12039–12053 (2017).
Johansen, J. et al. Evaluation of Tet-on system to avoid transgene down-regulation in ex vivo gene transfer to the CNS. Gene Ther. 9, 1291–1301 (2002).
Senturk, S. et al. Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization. Nat. Commun. 8, 1–10 (2017). 2017 81.
Aubrey, B. J. et al. An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo. Cell Rep. 10, 1422–1432 (2015).
Lin, S. et al. An in vivo CRISPR screening platform for prioritizing therapeutic targets in AML. Cancer Discov. 12, 432–449 (2021).
Knapp, D. J. H. F. et al. Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression. Nat. Commun. 10, 1–14 (2019).
Kiani, S. et al. CRISPR transcriptional repression devices and layered circuits in mammalian cells. Nat. Methods 11, 723–726 (2014).
Campa, C. C., Weisbach, N. R., Santinha, A. J., Incarnato, D. & Platt, R. J. Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Nat. Methods 16, 887–893 (2019).
Chylinski, K. et al. CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci. Nat. Commun. 10, 5454 (2019).
Mueller, S. et al. Evolutionary routes and KRAS dosage define pancreatic cancer phenotypes. Nature 554, 62–68 (2018).
Fomicheva, M. & Macara, I. G. Genome-wide CRISPR screen identifies noncanonical NF-κB signaling as a regulator of density-dependent proliferation. eLife 9, 1–24 (2020).
Panganiban, R. A. et al. Genome-wide CRISPR screen identifies suppressors of endoplasmic reticulum stress-induced apoptosis. Proc. Natl. Acad. Sci. USA. 116, 13384–13393 (2019).
Maddalo, D. et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516, 423–427 (2014).
Xu, C. L., Ruan, M. Z. C., Mahajan, V. B. & Tsang, S. H. Viral delivery systems for crispr. Viruses 11, 28 (2019).
Kaltenbacher, T. et al. CRISPR somatic genome engineering and cancer modelling in the mouse pancreas and liver. Nat. Protoc. 17, 1142–1188 (2022).
Ferrari, F. K., Samulski, T., Shenk, T. & Samulski, R. J. Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. J. Virol. 70, 3227–3234 (1996).
Maresch, R. et al. Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice. Nat. Commun. 7, 10770 (2016).
Liu, F., Song, Y. K. & Liu, D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 6, 1258–1266 (1999).
Yin, H. et al. Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat. Biotechnol. 35, 1179–1187 (2017).
Cheng, Q. et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing. Nat. Nanotechnol. 15, 313–320 (2020).
Fu, Z. & Xiang, J. Aptamer-functionalized nanoparticles in targeted delivery and cancer therapy. Int. J. Mol. Sci. 21, 1–39 (2020).
Liang, C. et al. Tumor cell-targeted delivery of CRISPR/Cas9 by aptamer-functionalized lipopolymer for therapeutic genome editing of VEGFA in osteosarcoma. Biomaterials 147, 68–85 (2017).
Platt, R. J. J. et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014).
Chu, V. T. et al. Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes. BMC Biotechnol. 16, 4 (2016).
Dow, L. E. et al. Conditional reverse tet-transactivator mouse strains for the efficient induction of tre-regulated transgenes in mice. PLoS ONE 9, e95236 (2014).
Katigbak, A., Robert, F., Paquet, M. & Pelletier, J. Inducible genome editing with conditional CRISPR/Cas9 mice. G3 Genes Genomes Genet. 8, 1627–1635 (2018).
Roper, J. et al. In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis. Nat. Biotechnol. 35, 569–576 (2017).
Feldser, D. M. et al. Stage-specific sensitivity to p53 restoration during lung cancer progression. Nature 468, 572–575 (2010).
MR, J. et al. Selective activation of p53-mediated tumour suppression in high-grade tumours. Nature 468, 567–571 (2010).
Rad, R. et al. A genetic progression model of BrafV600E-induced intestinal tumorigenesis reveals targets for therapeutic intervention. Cancer Cell 24, 15–29 (2013).
Rogers, Z. N. et al. Mapping the in vivo fitness landscape of lung adenocarcinoma tumor suppression in mice. Nat. Genet. 50, 483–486 (2018).
Cai, H. et al. A functional taxonomy of tumor suppression in oncogenic KRAS-driven lung cancer. Cancer Discov. 11, 1754–1773 (2021).
Yau, E. H. & Rana, T. M. Next-generation sequencing of genome-wide CRISPR screens. 1712, 203–216 (2018).
Canver, M. C. et al. Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments. Nat. Protoc. 13, 946–986 (2018).
Bodapati, S., Daley, T. P., Lin, X., Zou, J. & Qi, L. S. A benchmark of algorithms for the analysis of pooled CRISPR screens. Genome Biol. 21, 1–13 (2020).
Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).
Li, W. et al. Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR. Genome Biol. 16, 281 (2015).
Wang, B. et al. Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute. Nat. Protoc. 14, 756–780 (2019).
Hart, T. & Moffat, J. BAGEL: a computational framework for identifying essential genes from pooled library screens. BMC Bioinforma. 17, 164 (2016).
Daley, T. P. et al. CRISPhieRmix: a hierarchical mixture model for CRISPR pooled screens. Genome Biol. 19, 1–13 (2018).
Xu, C. et al. piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice. Proc. Natl. Acad. Sci. USA. 114, 722–727 (2017).
Henkel, L., Rauscher, B., Schmitt, B., Winter, J. & Boutros, M. Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library. BMC Biol. 18, 174 (2020).
Ozawa, T. & James, C. D. Establishing intracranial brain tumor xenografts with subsequent analysis of tumor growth and response to therapy using bioluminescence imaging. J. Vis. Exp. 41, e1986 (2010).
Meacham, C. E., Ho, E. E., Dubrovsky, E., Gertler, F. B. & Hemann, M. T. In vivo RNAi screening identifies regulators of actin dynamics as key determinants of lymphoma progression. Nat. Genet. 41, 1133–1137 (2009).
Braun, C. J. et al. Coordinated splicing of regulatory detained introns within oncogenic transcripts creates an exploitable vulnerability in malignant glioma. Cancer Cell 32, 411–426.e11 (2017).
Merino, D. et al. Barcoding reveals complex clonal behavior in patient-derived xenografts of metastatic triple negative breast cancer. Nat. Commun. 10, 1–12 (2019).
Carugo, A. et al. In vivo functional platform targeting patient-derived xenografts identifies WDR5–Myc association as a critical determinant of pancreatic cancer. Cell Rep. 16, 133–147 (2016).
Michlits, G. et al. CRISPR-UMI: single-cell lineage tracing of pooled CRISPR–Cas9 screens. Nat. Methods 14, 1191–1197 (2017).
Zhu, S. et al. Guide RNAs with embedded barcodes boost CRISPR-pooled screens. Genome Biol. 20, 20 (2019).
Morgens, D. W. et al. Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens. Nat. Commun. 8, 15178 (2017).
Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018).
Chen, C.-H. H. et al. Improved design and analysis of CRISPR knockout screens. Bioinformatics 34, 4095–4101 (2018).
Wegner, M. et al. Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome. eLife 8, 1–31 (2019).
Imkeller, K., Ambrosi, G., Boutros, M. & Huber, W. Gscreend: modelling asymmetric count ratios in CRISPR screens to decrease experiment size and improve phenotype detection. Genome Biol. 21, 53 (2020).
Farmer, H. et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917–921 (2005).
Bryant, H. E. et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434, 913–917 (2005).
Ashworth, A. & Lord, C. J. Synthetic lethal therapies for cancer: what’s next after PARP inhibitors? Nat. Rev. Clin. Oncol. 15, 564–576 (2018).
Horlbeck, M. A. et al. Mapping the genetic landscape of human cells. Cell 174, 953–967.e22 (2018).
Hegde, M., Strand, C., Hanna, R. E. & Doench, J. G. Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens. PLoS ONE 13, 1–16 (2018).
Hanna, R. E. & Doench, J. G. A case of mistaken identity. Nat. Biotechnol. 36, 802–804 (2018).
Diehl, V. et al. Minimized combinatorial CRISPR screens identify genetic interactions in autophagy. Nucleic Acids Res. 49, 5684–5704 (2021).
Najm, F. J. et al. Orthologous CRISPR–Cas9 enzymes for combinatorial genetic screens. Nat. Biotechnol. 36, 179–189 (2018).
DeWeirdt, P. C. et al. Optimization of AsCas12a for combinatorial genetic screens in human cells. Nat. Biotechnol. 39, 94–104 (2020).
Konermann, S. et al. Transcriptome engineering with RNA-targeting type VI–D CRISPR effectors. Cell 173, 665–676.e14 (2018).
Adamson, B. et al. A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response. Cell 167, 1867–1882.e21 (2016).
Dixit, A. et al. Perturb-seq: dissecting molecular circuits with scalable single-cell RNA profiling of pooled genetic screens. Cell 167, 1853–1866.e17 (2016).
Jaitin, D. A. et al. Dissecting immune circuits by linking CRISPR-pooled screens with single-cell RNA-seq. Cell 167, 1883–1896.e15 (2016).
Xie, S., Duan, J., Li, B., Zhou, P. & Hon, G. C. Multiplexed engineering and analysis of combinatorial enhancer activity in single cells. Mol. Cell 66, 285–299.e5 (2017).
Datlinger, P. et al. Pooled CRISPR screening with single-cell transcriptome readout. Nat. Methods 14, 297–301 (2017).
Kim, D. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat. Biotechnol. 34, 863–868 (2016).
Kleinstiver, B. P. et al. Genome-wide specificities of CRISPR–Cas Cpf1 nucleases in human cells. Nat. Biotechnol. 34, 869–874 (2016).
Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).
Chavez, A. et al. Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567 (2016).
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).
Kurt, I. C. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. 39, 41–46 (2021).
Hilton, I. B. et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015).
Xu, X. et al. A CRISPR-based approach for targeted DNA demethylation. Cell Discov. 2, 16009 (2016).
Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016).
Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015).
Wang, F. et al. Advances in CRISPR-Cas systems for RNA targeting, tracking and editing. Biotechnol. Adv. 37, 708–729 (2019).
Abudayyeh, O. O. et al. A cytosine deaminase for programmable single-base RNA editing. Science 365, 382–386 (2019).
Rudalska, R. et al. In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer. Nat. Med. 20, 1138–1146 (2014).
Weber, J. et al. CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice. Proc. Natl. Acad. Sci. USA. 112, 13982–13987 (2015).
Kim, M. J. & Ahituv, N. The hydrodynamic tail vein assay as a tool for the study of liver promoters and enhancers. Methods Mol. Biol. 1015, 279–289 (2013).
Hubner, E. K. et al. Constitutive and inducible systems for genetic in vivo modification of mouse hepatocytes using hydrodynamic tail vein injection. J. Vis. Exp. 2, 56613 (2018).
Sands, M. S. AAV-mediated liver-directed gene therapy. Methods Mol. Biol. 807, 141–157 (2011).
Wang, G. et al. Mapping a functional cancer genome atlas of tumor suppressors in mouse liver using AAV-CRISPR-mediated direct in vivo screening. Sci. Adv. 4, eaao5508 (2018).
Kim, M. et al. Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver. Sci. Adv. 7, eabf4398 (2021).
Ranzani, M. et al. Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer. Nat. Methods 10, 155–161 (2013).
Chiou, S. H. et al. Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing. Genes Dev. 29, 1576–1585 (2015).
Quirin, K. A. et al. Safety and efficacy of AAV retrograde pancreatic ductal gene delivery in normal and pancreatic cancer mice. Mol. Ther. Methods Clin. Dev. 8, 8–20 (2018).
DuPage, M., Dooley, A. L. & Jacks, T. Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase. Nat. Protoc. 4, 1064–1072 (2009).
Blasco, R. B. et al. Simple and rapid invivo generation of chromosomal rearrangements using CRISPR/Cas9 technology. Cell Rep. 9, 1219–1227 (2014).
Sanchez-Rivera, F. J. et al. Rapid modelling of cooperating genetic events in cancer through somatic genome editing. Nature 516, 428–431 (2014).
Limberis, M. P., Vandenberghe, L. H., Zhang, L., Pickles, R. J. & Wilson, J. M. Transduction efficiencies of novel AAV vectors in mouse airway epithelium in vivo and human ciliated airway epithelium in vitro. Mol. Ther. 17, 294–301 (2009).
Katz, M. G. et al. Targeted gene delivery through the respiratory system: rationale for intratracheal gene transfer. J. Cardiovasc. Dev. Dis. 6, 8 (2019).
Chow, M. Y. T., Chang, R. Y. K. & Chan, H.-K. Inhalation delivery technology for genome-editing of respiratory diseases. Adv. Drug Deliv. Rev. 168, 217–228 (2021).
Aschauer, D. F., Kreuz, S. & Rumpel, S. Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS ONE. 8, (2013).
Chow, R. D. et al. AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma. Nat. Neurosci. 20, 1329–1341 (2017).
Humbel, M. et al. Maximizing lentiviral vector gene transfer in the CNS. Gene Ther. 28, 75–88 (2021).
Passini, M. A. et al. Intraventricular brain injection of adeno-associated virus type 1 (AAV1) in neonatal mice results in complementary patterns of neuronal transduction to AAV2 and total long-term correction of storage lesions in the brains of β-glucuronidase-deficient mice. J. Virol. 77, 7034–7040 (2003).
Shinmyo, Y. et al. CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation OPEN. Sci. Rep. 6, 2061 (2016).
Klatt, D. et al. Competitive sgRNA screen identifies p38 MAPK as a druggable target to improve HSPC engraftment. Cells 9, 2194 (2020).
Pan, D. et al. Biodistribution and toxicity studies of VSVG-pseudotyped lentiviral vector after intravenous administration in mice with the observation of in vivo transduction of bone marrow. Mol. Ther. 6, 19–29 (2002).
Lang, J. F., Toulmin, S. A., Brida, K. L., Eisenlohr, L. C. & Davidson, B. L. Standard screening methods underreport AAV-mediated transduction and gene editing. Nat. Commun. 10, 3415 (2019).
Annunziato, S. et al. Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland. Genes Dev. 30, 1470–1480 (2016).
Wagner, S., Thresher, R., Bland, R. & Laible, G. Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk. Sci. Rep. 5, 15115 (2015).
Rubin, J. D., Nguyen, T. V., Allen, K. L., Ayasoufi, K. & Barry, M. A. Comparison of gene delivery to the kidney by adenovirus, adeno-associated virus, and lentiviral vectors after intravenous and direct kidney injections. Hum. Gene Ther. 30, 1559–1571 (2019).
Picconi, J. L. et al. Kidney-specific expression of GFP by in-utero delivery of pseudotyped adeno-associated virus 9. Mol. Ther. Methods Clin. Dev. 1, 14014 (2014).
Thai, H. B. D. et al. Kidney-Targeted cytosolic delivery of siRNA using a small-sized mirror DNA tetrahedron for enhanced potency. ACS Cent. Sci. 6, 2250–2258 (2020).
Hoa, O. et al. Imaging and manipulating pituitary function in the awake mouse. Endocrinology 160, 2271–2281 (2019).
Balmayor, E. R. & van Griensven, M. Gene therapy for bone engineering. Front. Bioeng. Biotechnol. 3, 9 (2015).
Madry, H., Orth, P. & Cucchiarini, M. Gene therapy for cartilage repair. Cartilage 2, 201–225 (2011).
Ain, Q. U., Campos, E. V. R., Huynh, A., Witzigmann, D. & Hedtrich, S. Gene delivery to the skin – how far have we come? Trends Biotechnol. 39, 474–487 (2021).
Acknowledgements
The authors apologize to colleagues whose work was not acknowledged owing to space limitations. C.J.B. is funded by the Max-Eder Program of Deutsche Krebshilfe (70113377) and The Care for Rare Foundation. D.S. is supported by the German Cancer Consortium (DKTK), Deutsche Forschungsgemeinschaft (DFG SA 1374/4-2, SFB 1321 Project-ID 329628492 P06, P11 and S01 and SFB 1371 Project-ID 395357507 P12) and the European Research Council (ERC CoG No. 648521). R.R. receives funds from the Deutsche Krebshilfe (70114314), the German Research Foundation (1629/4-1; SFB1321) and the European Research Council (Consolidator Grant 819642 PACA- MET and MSCA- ITN- ETN 861196).
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Braun, C. J. et al. Proc. Natl. Acad. Sci. USA. 113, E3892–E3900 (2016): https://doi.org/10.1073/pnas.1600582113
Maresch, R. et al. Nat. Commun. 7, 10770 (2016): https://doi.org/10.1038/ncomms10770
Weber, J. et al. Proc. Natl. Acad. Sci. USA. 112, 13982–13987 (2015): https://doi.org/10.1073/pnas.1512392112
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Braun, C.J., Adames, A.C., Saur, D. et al. Tutorial: design and execution of CRISPR in vivo screens. Nat Protoc 17, 1903–1925 (2022). https://doi.org/10.1038/s41596-022-00700-y
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DOI: https://doi.org/10.1038/s41596-022-00700-y
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