Abstract
Genome editing using the CRISPR/Cas9 RNA-guided endonuclease system has rapidly become a driving force for discovery in modern biomedical research. This simple yet elegant system has been widely used to generate both loss-of-function alleles and precision knock-in mutations using single-stranded donor oligonucleotides. Our CRISPRtools platform supports both of these applications in order to facilitate the use of CRISPR/Cas9. While there are several tools that facilitate CRISPR/Cas9 design and screen for potential off-target sites, the process is typically performed sequentially on single genes, limiting scalability for large-scale programs. Here, the design principle underlying gene ablation is based upon using paired guides flanking a critical region/exon of interest to create deletions. Guide pairs are rank ordered based upon published efficiency scores and off-target analyses, and reported in a concise format for downstream implementation. The exon deletion strategy simplifies characterization of founder animals and is the strategy employed for the majority of knockouts in the mouse. In proof-of-principle experiments, the effectiveness of this approach is demonstrated using microinjection and electroporation to introduce CRISPR/Cas9 components into mouse zygotes to delete critical exons.
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Acknowledgements
We would like to thank Charles Vejnar, Miguel Moreo-Mateos, and A. Giraldez at Yale University for kindly providing the CRISPRscan code; Susan Kales and Rachel Urban for technical support; Haoyi Wang and Wen-bo Wang for providing insight into electroporation experiments; and the Genetic Engineering Technologies and Transgenic Genotyping Services at the Jackson Laboratory. This work was supported by the Office Of The Director, National Institutes Of Health under Award Number OD011185 and UM1OD023222 (to S.A.M.), and OD010972 (to L.G.R.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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Peterson, K.A., Beane, G.L., Goodwin, L.O. et al. CRISPRtools: a flexible computational platform for performing CRISPR/Cas9 experiments in the mouse. Mamm Genome 28, 283–290 (2017). https://doi.org/10.1007/s00335-017-9681-z
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DOI: https://doi.org/10.1007/s00335-017-9681-z