Abstract
Genetically engineered mouse models (GEMMs) are very powerful tools to study lineage hierarchy and cellular dynamics of stem cells in vivo. Stem cell behavior in various contexts such as development, normal homeostasis and diseases have been investigated using GEMMs. The strategies to generate GEMMs have drastically changed in the last decade with the development of the CRISPR/Cas9 system for manipulation of the mammalian genome. The advantages of the CRISPR/Cas9 are its simplicity and efficiency. The bioinformatics tools available now allow us to quickly identify appropriate guide RNAs and design experimental conditions to generate the targeted mutation. In addition, the genome can be manipulated directly in the zygote which reduces the time to modify target genes compared to other technologies such as Embryonic Stem (ES) cells. Equally important is that we can manipulate the genome of any mouse background with the CRISPR/Cas9 system which omits time-consuming backcrossing processes, accelerates research and increases flexibility. Here, we will summarize basic allelic types and our standard strategies of how to generate them.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. https://doi.org/10.1126/science.1231143
Hilton IB, Gersbach CA (2015) Enabling functional genomics with genome engineering. Genome Res 25:1442–1455. https://doi.org/10.1101/gr.190124.115
Zuo E, Cai Y-J, Li K, Wei Y, Wang B-A, Sun Y, Liu Z, Liu J, Hu X, Wei W, Huo X, Shi L, Tang C, Liang D, Wang Y, Nie Y-H, Zhang C-C, Yao X, Wang X, Zhou C, Ying W, Wang Q, Chen R-C, Shen Q, Xu G-L, Li J, Sun Q, Xiong Z-Q, Yang H (2017) One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs. Cell Res 27:933–945. https://doi.org/10.1038/cr.201
Lieber MR (2010) The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 79:181–211. https://doi.org/10.1146/annurev.biochem.052308.093131
Casola S (2010) Mouse models for miRNA expression: the ROSA26 locus. In: Monticelli S (ed) MicroRNAs and the immune system. Humana Press, Totowa, NJ, pp 145–163
Gu B, Posfai E, Rossant J (2018) Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat Biotechnol 36:632–637. https://doi.org/10.1038/nbt.4166
Miura H, Quadros RM, Gurumurthy CB, Ohtsuka M (2018) Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat Protoc 13:195–215. https://doi.org/10.1038/nprot.2017.153
Yao X, Zhang M, Wang X, Ying W, Hu X, Dai P, Meng F, Shi L, Sun Y, Yao N, Zhong W, Li Y, Wu K, Li W, Chen Z, Yang H (2018) Tild-CRISPR allows for efficient and precise gene knockin in mouse and human cells. Dev Cell 45:526–536.e5. https://doi.org/10.1016/j.devcel.2018.04.021
Behringer R, Gertsenstein M, Nagy KV, Nagy A (2014) Manipulating the mouse embryo: a laboratory manual, 4th edn. Cold Spring Harbor, New York
Yamanaka Y (2016) CRISPR/Cas9 genome editing as a strategy to study the tumor microenvironment in transgenic mice. In: Ursini-Siegel J, Beauchemin N (eds) The tumor microenvironment. Springer, New York, pp 261–271
Posfai E, Petropoulos S, de Barros FRO, Schell JP, Jurisica I, Sandberg R, Lanner F, Rossant J (2017) Position- and Hippo signaling-dependent plasticity during lineage segregation in the early mouse embryo. elife 6:e22906. https://doi.org/10.7554/eLife.22906
Ohtsuka M, Sato M, Miura H, Takabayashi S, Matsuyama M, Koyano T, Arifin N, Nakamura S, Wada K, Gurumurthy CB (2018) i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases. Genome Biol 19:25. https://doi.org/10.1186/s13059-018-1400-x
Whitten WK (1956) Modification of the oestrous cycle of the mouse by external stimuli associated with the male. J Endocrinol 13:399–404. https://doi.org/10.1677/joe.0.0130399
Pollock JD (1996) Mouse genetics: concepts and applications. In: Silver LM (ed) The quarterly review of biology, vol 71, pp 123–123. https://doi.org/10.1086/419294
Liang X, Potter J, Kumar S, Ravinder N, Chesnut JD (2017) Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA. J Biotechnol 241:136–146. https://doi.org/10.1016/j.jbiotec.2016.11.011
Haeussler M, Schönig K, Eckert H, Eschstruth A, Mianné J, Renaud J-B, Schneider-Maunoury S, Shkumatava A, Teboul L, Kent J, Joly J-S, Concordet J-P (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17:148. https://doi.org/10.1186/s13059-016-1012-2
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Desjardins, J., Cowan, M., Yamanaka, Y. (2022). Designing Genetically Engineered Mouse Models (GEMMs) Using CRISPR Mediated Genome Editing. In: Kannan, N., Beer, P. (eds) Stem Cell Assays. Methods in Molecular Biology, vol 2429. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1979-7_36
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1979-7_36
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1978-0
Online ISBN: 978-1-0716-1979-7
eBook Packages: Springer Protocols