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Mammalian Reverse Genetics without Crossing Reveals Nr3a as a Short-Sleeper Gene

2016; Cell Press; Volume: 14; Issue: 3 Linguagem: Inglês

10.1016/j.celrep.2015.12.052

2639-1856

Genshiro A. Sunagawa, Kenta Sumiyama, Maki Ukai‐Tadenuma, Dimitri Perrin, Hiroshi Fujishima, Hideki Ukai, Osamu Nishimura, Shoi Shi, Rei-ichiro Ohno, Ryohei Narumi, Yoshihiro Shimizu, Daisuke Tone, Koji L. Ode, Shigehiro Kuraku, Hiroki R. Ueda,

Genetics, Aging, and Longevity in Model Organisms

The identification of molecular networks at the system level in mammals is accelerated by next-generation mammalian genetics without crossing, which requires both the efficient production of whole-body biallelic knockout (KO) mice in a single generation and high-performance phenotype analyses. Here, we show that the triple targeting of a single gene using the CRISPR/Cas9 system achieves almost perfect KO efficiency (96%-100%). In addition, we developed a respiration-based fully automated non-invasive sleep phenotyping system, the Snappy Sleep Stager (SSS), for high-performance (95.3% accuracy) sleep/wake staging. Using the triple-target CRISPR and SSS in tandem, we reliably obtained sleep/wake phenotypes, even in double-KO mice. By using this system to comprehensively analyze all of the N-methyl-D-aspartate (NMDA) receptor family members, we found Nr3a as a short-sleeper gene, which is verified by an independent set of triple-target CRISPR. These results demonstrate the application of mammalian reverse genetics without crossing to organism-level systems biology in sleep research.