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Cell Rep. 2016 Jan 26;14(3):662-677. doi: 10.1016/j.celrep.2015.12.052. Epub 2016 Jan 7.

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

Author information

1
Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
2
Laboratory for Mouse Genetic Engineering, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
3
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.
4
Phyloinformatics Unit, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
5
Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
6
Laboratory for Cell-Free Protein Synthesis, RIKEN Quantitative Biology Center, 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan.
7
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.

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.

PMID:
26774482
DOI:
10.1016/j.celrep.2015.12.052
[Indexed for MEDLINE]
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