One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering

The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances. [Display omitted] •One-step generation of mice with reporters in endogenous genes•One-step generation of conditional mutant mice•Off-target analysis suggests high specificity of the CRISPR/Cas9 system CRISPR/Cas technology is used for insertion of DNA reporter constructs into endogenous genes, derivation of conditional mutant mice and generation of deletions of defined length. Off-target mutations appear only in rare occasions.