CRISPRtools: a flexible computational platform for performing CRISPR/Cas9 experiments in the mouse.
Mamm Genome 2017 Mar 9. [Epub ahead of print]
OD011185, OD023222, OD010972
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.
Peterson, Kevin A; Beane, Glen L; Goodwin, Leslie O; Kutny, Peter M; Reinholdt, Laura G; and Murray, Stephen A., "CRISPRtools: a flexible computational platform for performing CRISPR/Cas9 experiments in the mouse." (2017). Faculty Research Ahead of Print. 12.