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Mamm Genome. 2017 Aug;28(7-8):283-290. doi: 10.1007/s00335-017-9681-z. Epub 2017 Mar 9.

CRISPRtools: a flexible computational platform for performing CRISPR/Cas9 experiments in the mouse.

Author information

1
The Jackson Laboratory, 600 Main St., Bar Harbor, ME, 04609, USA.
2
The Jackson Laboratory, 600 Main St., Bar Harbor, ME, 04609, USA. Steve.Murray@jax.org.

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.

KEYWORDS:

Cas9 Protein; Efficiency Score; Exon Deletion; Genome Editing; Nonsense Mediate Decay

PMID:
28280930
PMCID:
PMC5591755
DOI:
10.1007/s00335-017-9681-z
[Indexed for MEDLINE]
Free PMC Article

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