title: A mouse geneticist's practical guide to CRISPR applications.
abstract: CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Even for the laboratory mouse, a model that has thrived under the benefits of embryonic stem (ES) cell knockout capabilities for nearly three decades, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 technology enables one to manipulate the genome with unprecedented simplicity and speed. It allows generation of null, conditional, precisely mutated, reporter, or tagged alleles in mice. Moreover, it holds promise for other applications beyond genome editing. The crux of this system is the efficient and targeted introduction of DNA breaks that are repaired by any of several pathways in a predictable but not entirely controllable manner. Thus, further optimizations and improvements are being developed. Here, we summarize current applications and provide a practical guide to use the CRISPR/Cas9 system for mouse mutagenesis, based on published reports and our own experiences. We discuss critical points and suggest technical improvements to increase efficiency of RNA-guided genome editing in mouse embryos and address practical problems such as mosaicism in founders, which complicates genotyping and phenotyping. We describe a next-generation sequencing strategy for simultaneous characterization of on- and off-target editing in mice derived from multiple CRISPR experiments. Additionally, we report evidence that elevated frequency of precise, homology-directed editing can be achieved by transient inhibition of the Ligase IV-dependent nonhomologous end-joining pathway in one-celled mouse embryos.
authors.affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, New York 14850.
authors.forename: Priti
authors.initials: P
authors.lastname: Singh
authors.affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, New York 14850 jcs92@cornell.edu.
authors.forename: John C
authors.initials: JC
authors.lastname: Schimenti
authors.affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, New York 14850.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Bolcun-Filas
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D018703:Founder Effect; D018390:Gene Targeting / Q000379:methods*; D060005:Genotyping Techniques; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*
keywords: CRISPR; genome editing; mouse knockouts; nonhomologous end joining