title: Mammalian Reverse Genetics without Crossing Reveals Nr3a as a Short-Sleeper Gene.
abstract: 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.
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Genshiro A
authors.initials: GA
authors.lastname: Sunagawa
authors.affiliation: Laboratory for Mouse Genetic Engineering, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Sumiyama
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Maki
authors.initials: M
authors.lastname: Ukai-Tadenuma
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan; School of Electrical Engineering and Computer Science, Science and Engineering Faculty, Queensland University of Technology, GPO Box 2434, Brisbane, QLD 4001, Australia.
authors.forename: Dimitri
authors.initials: D
authors.lastname: Perrin
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fujishima
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Ukai
authors.affiliation: Phyloinformatics Unit, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Nishimura
authors.affiliation: Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Shoi
authors.initials: S
authors.lastname: Shi
authors.affiliation: Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Rei-Ichiro
authors.initials: RI
authors.lastname: Ohno
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Narumi
authors.affiliation: Laboratory for Cell-Free Protein Synthesis, RIKEN Quantitative Biology Center, 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Shimizu
authors.affiliation: Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Tone
authors.affiliation: Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Koji L
authors.initials: KL
authors.lastname: Ode
authors.affiliation: Phyloinformatics Unit, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
authors.forename: Shigehiro
authors.initials: S
authors.lastname: Kuraku
authors.affiliation: Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan; Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. Electronic address: uedah-tky@umin.ac.jp.
authors.forename: Hiroki R
authors.initials: HR
authors.lastname: Ueda
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics; D004569:Electroencephalography; D004576:Electromyography; D005260:Female; D005838:Genotype; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D014442:Monophenol Monooxygenase / Q000172:deficiency / Q000235:genetics; D010641:Phenotype; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics* / Q000378:metabolism; D059386:Reverse Genetics; D012890:Sleep / Q000502:physiology*; D014851:Wakefulness / Q000502:physiology*
chemical_list: C464437:NR3A NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D014442:Monophenol Monooxygenase