Chapter 18 - Genetic modification of mice using CRISPR-Cas9: Best practices and practical concepts explained
Document Type
Article
Publication Date
2024
Original Citation
Hosur V,
Low BE,
Wiles MV.
Chapter 18 - Genetic modification of mice using CRISPR-Cas9: Best practices and practical concepts explained Rigor and Reproducibility in Genetics and Genomics: Academic Press; 2024. p. 425-52.
JAX Source
Rigor and Reproducibility in Genetics and Genomics: Academic Press; 2024. p. 425-52.
DOI
https://doi.org/10.1016/B978-0-12-817218-6.00018-8
Grant
We acknowledge support from the National Institutes of Health under Award Number R01 CA265978 (VH), and the Director’s Innovation Fund at JAX (JAX-DIF-FY17).
Abstract
The development of precision targetable nucleases has led to a massive acceleration in creating ge- netically modified mice and other species [1–6]. This began more than a decade ago with Zinc Finger Nucleases (ZFNs) [7], followed by TALENs (Transcription Activator-Like Effector Nucleases) [8–10], and then CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats with CRISPR- associated effector protein 9) [11,12]. At present, RNA-guided CRISPR-Cas9 is the most affordable and straightforward to design, construct, and implement. This accessibility, combined with its gener- ally high degree of targeting efficiency, has pushed CRISPR-Cas9 to the forefront of gene-editing methods. Regardless of its relative simplicity, the complexity of the resulting nuclease-derived genetic modifications, including the modified organism’s phenotype, should not be underestimated [13–15].
Herein, we outline our experience using CRISPR-Cas9 to precisely and directly engineer mouse zygotes, focusing on the general methodology and screening used to characterize the resulting alleles. These screening strategies are simple, straightforward, and reproducible. While the focus of this chap- ter is on CRISPR-modified alleles generated in mice, these screening regimes can be applied to other organisms and to the characterization of genetic modifications resulting from ZFNs, TALENs, or any other gene-editing technology.