title: CRISPRtools: a flexible computational platform for performing CRISPR/Cas9 experiments in the mouse.
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.
authors.affiliation: The Jackson Laboratory, 600 Main St., Bar Harbor, ME, 04609, USA.
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Peterson
authors.affiliation: The Jackson Laboratory, 600 Main St., Bar Harbor, ME, 04609, USA.
authors.forename: Glen L
authors.initials: GL
authors.lastname: Beane
authors.affiliation: The Jackson Laboratory, 600 Main St., Bar Harbor, ME, 04609, USA.
authors.forename: Leslie O
authors.initials: LO
authors.lastname: Goodwin
authors.affiliation: The Jackson Laboratory, 600 Main St., Bar Harbor, ME, 04609, USA.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Kutny
authors.affiliation: The Jackson Laboratory, 600 Main St., Bar Harbor, ME, 04609, USA.
authors.forename: Laura G
authors.identifier: http://orcid.org/0000-0002-0594-1702
authors.initials: LG
authors.lastname: Reinholdt
authors.affiliation: The Jackson Laboratory, 600 Main St., Bar Harbor, ME, 04609, USA. Steve.Murray@jax.org.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Murray
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D019295:Computational Biology / Q000379:methods*; D005091:Exons; D000072669:Gene Editing / Q000379:methods; D060005:Genotyping Techniques; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D059365:Nonsense Mediated mRNA Decay; D000094704:RNA, Guide, CRISPR-Cas Systems; D017384:Sequence Deletion; D012984:Software; D064878:Web Browser; D057188:Workflow; D015053:Zygote
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: Cas9 Protein; Efficiency Score; Exon Deletion; Genome Editing; Nonsense Mediate Decay