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

• Main Authors: 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
• Format: Article
• Language: English
• Published: Elsevier 2016-01-01
• Series: Cell Reports
• Online Access: http://www.sciencedirect.com/science/article/pii/S221112471501493X

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.