CRISPR/Cas9-Mediated Insertion of loxP Sites in the Mouse Dock7 Gene Provides an Effective Alternative to Use of Targeted Embryonic Stem Cells.

摘要: Targeted gene mutation in the mouse is a primary strategy to understand function and relation phenotype. The Knockout Mouse Project (KOMP) had an initial goal develop public resource of embryonic stem (ES) cell clones that carry null mutations all genes. Indeed, many useful novel models have been generated from publically accessible targeted ES lines. However, there are limitations, including incorrect targeting or cassette structure, difficulties with germline transmission allele chimeric mice. In our experience, using small sample clones, we were successful ∼50% time generating correctly allele. With advent CRISPR/Cas9 as genome modification tool, assessed efficiency creating conditional one gene, dedicator cytokinesis 7 (Dock7), for which unsuccessful KOMP clone. was insert loxP sites flank either exons 3 4, through 7. By coinjecting Cas9 mRNA, validated sgRNAs, oligonucleotide donors into fertilized eggs C57BL/6J mice, obtained variety alleles, mice homozygous alleles mediated by nonhomologous end joining, two desired sites, both sites. We also found frequent inserted sequence, partly attributable heterogeneity original preparation.