Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR–Cas9-mediated mutagenesis

摘要: Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9 system, allow simple and rapid genetic modification in most model organisms human cell lines. Here, we report production analysis of mice carrying inactivation via deletion a genomic insulator, key non-coding regulatory DNA element found 5′ upstream mouse tyrosinase (Tyr) gene. Targeting sequences flanking this boundary fertilized eggs resulted efficient or inversion large intervening fragments delineated by RNA guides. The resulting genome-edited showed dramatic decrease Tyr gene expression inferred from evident coat pigmentation, thus supporting functionality sequence vivo, at endogenous locus. Several potential off-targets bearing similarity with each two guides used were analyzed to be largely intact. This study reports how elements, even if located repeat-rich sequences, can efficiently functionally evaluated vivo and, furthermore, it illustrates elements described ENCODE EPIGENOME projects, genomes, systematically validated.