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Tutorial: design and execution of CRISPR in vivo screens

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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Fig. 1: In vivo screening phenotypes and examples of focused libraries.
Fig. 2: The spectrum of CRISPR system applications in eukaryotes.
Fig. 3: Engineered CRISPR systems for inducing one or more perturbations per cell.
Fig. 4: Stages of a transplantation-based CRISPR in vivo screen.
Fig. 5: Features of polymerase II and III promoters.
Fig. 6: Systems for the delivery of CRISPR components into living tissues.
Fig. 7: Spatial and temporal control of CRISPR activity in direct in vivo screens.
Fig. 8: NGS-based strategies for the analysis of pooled in vivo CRISPR screens.
Fig. 9: Exemplary results: Transplantation-based CRISPRa screen for genetic mediators of bone marrow relapse in leukemia.
Fig. 10: Various bottlenecks during a transplantation-based in vivo screen impact library representation and lead to the dominance of strong enrichers.

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).

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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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C.J.B., A.C.A., D.S. and R.R. all researched data for the article, provided a substantial contribution to discussions of the content and contributed to writing the article and to the review and/or editing of the manuscript before submission.

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Correspondence to Christian J. Braun or Roland Rad.

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Key references

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 (2022). https://doi.org/10.1038/s41596-022-00700-y

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