title: Detailed phenotypic and molecular analyses of genetically modified mice generated by CRISPR-Cas9-mediated editing.
abstract: The bacterial CRISPR-Cas9 system has been adapted for use as a genome editing tool. While several recent reports have indicated that successful genome editing of mice can be achieved, detailed phenotypic and molecular analyses of the mutant animals are limited. Following pronuclear micro-injection of fertilized eggs with either wild-type Cas9 or the nickase mutant (D10A) and single or paired guide RNA (sgRNA) for targeting of the tyrosinase (Tyr) gene, we assessed genome editing in mice using rapid phenotypic readouts (eye and coat color). Mutant mice with insertions or deletions (indels) in Tyr were efficiently generated without detectable off-target cleavage events. Gene correction of a single nucleotide by homologous recombination (HR) could only occur when the sgRNA recognition sites in the donor DNA were modified. Gene repair did not occur if the donor DNA was not modified because Cas9 catalytic activity was completely inhibited. Our results indicate that allelic mosaicism can occur following -Cas9-mediated editing in mice and appears to correlate with sgRNA cleavage efficiency at the single-cell stage. We also show that larger than expected deletions may be overlooked based on the screening strategy employed. An unbiased analysis of all the deleted nucleotides in our experiments revealed that the highest frequencies of nucleotide deletions were clustered around the predicted Cas9 cleavage sites, with slightly broader distributions than expected. Finally, additional analysis of founder mice and their offspring indicate that their general health, fertility, and the transmission of genetic changes were not compromised. These results provide the foundation to interpret and predict the diverse outcomes following CRISPR-Cas9-mediated genome editing experiments in mice.
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, 63110, United States of America.
authors.forename: Bijal A
authors.initials: BA
authors.lastname: Parikh
authors.affiliation: Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, 63110, United States of America.
authors.forename: Diana L
authors.initials: DL
authors.lastname: Beckman
authors.affiliation: Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, 63110, United States of America.
authors.forename: Swapneel J
authors.initials: SJ
authors.lastname: Patel
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, 63110, United States of America.
authors.forename: J Michael
authors.initials: JM
authors.lastname: White
authors.affiliation: Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, 63110, United States of America; Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO, 63110, United States of America.
authors.forename: Wayne M
authors.initials: WM
authors.lastname: Yokoyama
mesh_terms: D000483:Alleles; D000818:Animals; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D016678:Genome; D051379:Mice; D008822:Mice, Transgenic; D014442:Monophenol Monooxygenase / Q000235:genetics*; D010641:Phenotype; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D014442:Monophenol Monooxygenase