Article

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

Authors
Genshiro A. Sunagawa, Kenta Sumiyama,
Maki Ukai-Tadenuma, Dimitri Perrin, ...,
Koji L. Ode, Shigehiro Kuraku,
Hiroki R. Ueda

Correspondence
uedah-tky@umin.ac.jp

In Brief
Sunagawa et al. present an application of
mammalian reverse genetics withoutcrossing by developing two methods. The
authors improve KO mice production
using a triple-target CRISPR and combine
this with a non-invasive respirationbased automated sleep phenotyping
system, the Snappy Sleep Stager.
Combining these methods, the authors
found that the Nr3a KO is a short-sleeper.

HighlightsdA triple-target CRISPR method achieved almost perfectknockout efficiencyd

SSS achieved non-invasive fully automated highperformance sleep phenotypingdGenetics without crossing revealed Nr3a as a short-sleeper
gene

Sunagawa et al., 2016, Cell Reports 14, 1–16
January 26, 2016 ª2016 The Authors
http://dx.doi.org/10.1016/j.celrep.2015.12.052

Please cite this article in press as: Sunagawa et al., Mammalian Reverse Genetics without Crossing Reveals Nr3a as a Short-Sleeper Gene, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2015.12.052

Cell Reports

ArticleMammalian Reverse Genetics without Crossing
Reveals Nr3a as a Short-Sleeper Gene
Genshiro A. Sunagawa,1,8 Kenta Sumiyama,2,8 Maki Ukai-Tadenuma,1,8 Dimitri Perrin,1,6,8 Hiroshi Fujishima,1
Hideki Ukai,1 Osamu Nishimura,5 Shoi Shi,3 Rei-ichiro Ohno,3 Ryohei Narumi,1 Yoshihiro Shimizu,7 Daisuke Tone,3
Koji L. Ode,3 Shigehiro Kuraku,5 and Hiroki R. Ueda1,3,4,*
1Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
2Laboratory for Mouse Genetic Engineering, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
3Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
113-0033, Japan
4CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
5Phyloinformatics Unit, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo
650-0047, Japan
6School of Electrical Engineering and Computer Science, Science and Engineering Faculty, Queensland University of Technology, GPO Box
2434, Brisbane, QLD 4001, Australia
7Laboratory for Cell-Free Protein Synthesis, RIKEN Quantitative Biology Center, 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan
8Co-first author*Correspondence: uedah-tky@umin.ac.jp
http://dx.doi.org/10.1016/j.celrep.2015.12.052
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

SUMMARY

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-bodybiallelic knockout (KO) mice in a single generationand high-performance phenotype analyses. Here,
we show that the triple targeting of a single geneusing the CRISPR/Cas9 system achieves almost
perfect KO efficiency (96%–100%). In addition, wedeveloped a respiration-based fully automated noninvasive 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 allof 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 applicationof mammalian reverse genetics without crossing toorganism-level systems biology in sleep research.
INTRODUCTION
The system-level identification of molecular networks in organisms is an important challenge in biology (Kitano, 2002a,
2002b). Classical reverse genetics requires several generationsof animal crosses to produce mutant animals of sufficient quality
and quantity for phenotype analysis. Conventional methods for
producing knockout (KO) mice usually involve targeting-vector

construction (2 weeks), the introduction of target mutations
into embryonic stem cells (ESCs) by homologous recombination(a few weeks), and the injection of the mutant ESCs into wildtype blastocysts to produce chimera mice (3 weeks). If themutant ESCs contribute to the germline of the newborn chimera
mice, their next-generation offspring will possess a heterozygous mutation (3 months). Further crossings of the offspring(several months to years; at least 3 months per generation) willproduce mice with completely homozygous KO mutations on
an inbred genomic background, which is required for reliablephenotype analysis. Thus, conventional methods require substantial amounts of time, space, and effort to knock out even asingle gene (Nagy, 2003). Therefore, identification of molecular
networks comprehensively in organisms will require ‘‘next-generation’’ genetics, i.e., genetic alterations without crossing. Toward this goal, both highly efficient (>90%) mutant production
in a single generation and highly accurate (>90%) phenotype analyses of mutant animals are needed.
The efficient production of biallelic KO mice can be facilitatedby recently developed genome editing techniques, including
ZFNs (zinc-finger nucleases) (Carbery et al., 2010), TALENs (transcription-activator-like effector nucleases) (Sung et al., 2013),
and the CRISPR/Cas nuclease systems (Hsu et al., 2014; Wanget al., 2013). Unlike the protein-based ZFNs and TALENs, the
CRISPR/Cas system uses RNA-based DNA recognition derived
from a bacterial adaptive immune system (Horvath and Barrangou, 2010; Wiedenheft et al., 2012). This method accelerates
the generation of KO animals via the co-injection of RNA encoding the Cas9 protein and target-locus-specific guide RNAs(gRNAs) into embryos (Fujii et al., 2013; Wang et al., 2013).
Several modifications of the CRISPR/Cas9 system have been
also introduced to improve the efficiency and specificity of targeted mutations in a genome (Fu et al., 2014; Ran et al., 2013;
Zhou et al., 2014). However, two problems remain: (1) first-generation mice often contain a mosaic of wild-type and KO cells, andCell Reports 14, 1–16, January 26, 2016 ª2016 The Authors 1

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Please cite this article in press as: Sunagawa et al., Mammalian Reverse Genetics without Crossing Reveals Nr3a as a Short-Sleeper Gene, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2015.12.052(2) the rate of complete biallelic mutant mice generated is relatively low (usually 60%–80% at best). Therefore, the highly efficient (>90%) production of whole-body biallelic KO in a single
generation remains a fundamental challenge for organism-levelsystems biology.
The comprehensive identification of molecular circuits at theorganism level also requires accurate (>90%) phenotype analysis. In neuroscience, sleep/wake behavior is an intriguing
phenotype, because sleep disorders (e.g., insomnia or hypersomnia) are sensitive and informative symptoms of almost all
psychological disorders. Sleep/wake states have been characterized in humans by electroencephalography (EEG) and electromyography (EMG). For example, during sleep, EEG mostly
displays high-amplitude, low-frequency fluctuations, whereas
during waking, it exhibits low-amplitude, high-frequency fluctuations. On the other hand, EMG displays low-amplitude fluctuations during sleep and high-amplitude fluctuations during
waking. These characteristic EEG/EMG patterns during sleep
and waking are preserved in mammals and can be measured
by electrodes surgically implanted in the brain and muscles.
However, such recording requires special surgical skills, andthe surgery is highly invasive, requiring a long recovery period(more than 10 days) after implantation before sleep/wakerecording. Furthermore, the EEG/EMG data are often manuallyannotated and classified into sleep/wake phenotypes by visualassessment, which can be time consuming and somewhat
subjective. Therefore, sleep/wake phenotyping has been alow-throughput method; for comprehensive studies, a scalable,
non-invasive, fully automated sleep/wake recording method isneeded.
In this study, we developed a simple theory to predict theminimum efficacy of different CRISPR methods for the highly
efficient production of whole-body biallelic KO mice. As
we predicted, triple-target CRISPR elicited almost perfect(96%–100%) whole-body KO of the Tyrosinase (Tyr) gene,which is functionally evaluated by animal coat color. This KO efficiency was confirmed using three independent sets of gRNAs.
The highly efficient production of whole-body KO by the tripletarget CRISPR method also enabled us to obtain clock mutant
phenotypes reliably, not only of Bmal1 single-KO mice but alsoof Cry1/Cry2 or Per1/Per2 double-KO mice. For accurate phenotype analysis, we developed a respiration-based, non-invasive,
fully automated system, the Snappy Sleep Stager (SSS), whichenabled the high-performance analysis (95.3% accuracy) of
sleep/wake phenotypes. Using the triple-target CRISPR and
SSS methods, we analyzed the N-methyl-D-aspartate (NMDA)
receptor (NMDAR) family comprehensively and found that the
Nr3a KO mouse is a short sleeper (96.7 min/day less than wildtype). Finally, we developed a freely accessible web-based database that contains the triple-target candidates for 81.2% of thegenes in the mouse genome.
RESULTS
Triple-Target CRISPR Achieves Almost Perfect KO
Efficiency
To obtain a highly efficient method for producing biallelic KO
mice, we first constructed a simple computational model to es-

timate the minimum efficacy of different CRISPR methods (Supplemental Experimental Procedures). According to this computational model, a multiple-target CRISPR strategy, in which
multiple gRNAs target the same gene, is more efficient thanthe high-concentration CRISPR strategy, in which a multiplefold higher concentration of a single gRNA targets the gene of interest (Figures 1A and 1B; Figures S1A and S1B). This finding is
consistent with a previous report on dual-target CRISPR (Zhouet al., 2014). Our model further predicted that triple-target
CRISPR would achieve 79%–97% efficiency for the biallelic
KO of a single gene (Figure S1C). Importantly, if it achieves
more than 90% efficiency for the biallelic KO of a single gene,it can also achieve more than 80% efficiency even for the biallelic
KO of dual genes, which is sufficient for reliable analysis ofbehavioral phenotypes.
To test the model’s predictions, we selected Tyr as a target
gene and C57BL/6N as a basal inbred strain, because if thisgene is knocked out biallelically, the black coat color of
C57BL/6N becomes white. We designed three gRNAs for threetarget sequences of the Tyr gene (gRNA1, gRNA2, and gRNA3
for targets Tyr-1, Tyr-2, and Tyr-3, respectively; Figures 1C
and S1D). We confirmed that the designed gRNAs had highcleavage efficiency (7-fold to 40-fold compared to control)at least in cellulo by using a single strand annealing (SSA) assay(Figures 1D and S1E), in which the DNA cleavage of a target
sequence by the CRISPR/Cas9 system induced the recombination of incomplete fragments of firefly Luciferase (Luc), resulting
in an entire Luc gene and enhanced bioluminescence.
We then estimated two parameters of the model, an effective
total gRNA concentration compared to the dissociation constantof gRNA (S) and a recovery rate of a mutation (a). When injected
into fertilized eggs, the single-target strategy using each of thethree gRNAs alone at 150 ng/ml exhibited only moderate efficacy(36.0%, 54.2%, and 64.7%, for gRNA1, gRNA2, and gRNA3,
respectively; Figures 1F and S1G). Based on these results, S
and a were estimated to be 5.72 and 0.136, respectively. This allowed us to predict the minimum efficacy of the triple-target
CRISPR method for whole-body biallelic KO mice as 82.6%(Figure S1F). In fact, the triple-target strategy using the mixtureof Cas9 mRNA (100 ng/ml) and three gRNAs (gRNA1, gRNA2,
and gRNA3, each at 50 ng/ml) achieved almost perfect efficiency(97.5%; Figures 1E and 1F). We also performed qPCR of thetarget region including the CRISPR-target sequence and confirmed that five out of six CRISPR-target regions from two Tyr
KO mice were not detectable. These qPCR results matchedwith each other among the genomic DNAs extracted from brains,
scalps, and tails (Figure S1H), which is consistent with theobserved whole-body biallelic KO phenotype (i.e., white coat color) of the Tyr gene. We noted that observed KO efficacy (97.5%)
is more than the predicted minimum efficiency (82.6%) (Figure S1F). We speculated that the increased KO efficacy would
be because of irreversible mutations such as deletion between
multiple CRISPR target sites.
To investigate its genomic effect, we performed exome
sequencing; results were that 91.6%–95.6% of target bases
were covered more than ten times (Table S5). As a result, it
demonstrated on-target deletions of genomic stretches with variable lengths (Figure 2) and no off-target mutation. Thus, the2 Cell Reports 14, 1–16, January 26, 2016 ª2016 The Authors
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gRNA-1 gRNA-2

gRNA-3B

Cytoplasmic
Injection

α = 0.10
Single-allelic knockout
1.0

Gene

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Gene

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A

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10
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Tyr target sequence-3
gaagaagaagcaaccccagg (+)

Tyr-2

Tyr target sequence-2
gttattgctgcagctctctc (+)

20

Tyr-3

Tyr exon530(-)

Tyr target sequence-1
gtgtcaagggacacactgct (+)

40

pX330

Tyr exon1

50

Tyr-1

87,430,000

(-)

-1

87,460,000

pX330

chr7 (-) 87,490,000

Relative Luc Activity

C

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Total gRNA concentration (S)pSSA-Tyr-1 pSSA-Tyr-2/3

E

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Triple target (Tyr-1,2,3)Single(Tyr-1)

Single(Tyr-2)Single(Tyr-3)

Triple(Tyr-1,2,3)gRNA1(ng/µl)

150

0

0

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gRNA2
(ng/µl)

0

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(ng/µl)

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Embryos

73

58

198

251

Pups

25

24

34

40

Albino9(36.0%)

13(54.2%)

22(64.7%)

39(97.5%)

Pigmented

16(64.0%)

11(45.8%)

12(35.3%)

1(2.5%)

Figure 1. A Triple-Target CRISPR Method for Highly Efficient Production of Whole-Body Biallelic KO Mice(A) Schematic diagram of triple-target and single-target CRISPR methods. Each method uses the same amount of total gRNA.
(B) Computational simulation predicting how efficiently one or more alleles are cut out when different numbers of gRNAs are used against the same target gene. The
triple-target (red line) and single-target (blue line) methods were compared. The line and shaded area around the line indicate the mean and the SD of 1,000 simulations, respectively. The difference in efficiency between triple-target and single-target strategies became more apparent as the number of target alleles increased.
(C) Target sequences of the gRNAs for KO of the Tyrosinase (Tyr) gene. The short colored bars (blue, orange, and green) indicate the 20-base target sequences.
The target sequences were on the sense strand of the genomic DNA (plus sign refers to the gene’s sense strand).
(D) SSA assay for the gRNAs of the Tyr gene. Relative luciferase activities from the SSA vectors (pSSA-Tyr-1 and pSSA-Tyr-2) were measured. The SSA vector
was transfected into 293T cells with empty vector (-), pX330 Cas9 vector without any gRNA, or pX330 Cas9 and Tyr gRNA (Tyr-1, Tyr-2, or Tyr-3). The relative
Luciferase activity for each sample was scaled so that the activity with empty vector (-) was defined as 1. Error bars represent SD (n = 3).
(E) Tyr KO mice created by the triple-target CRISPR method. The coat color showed the biallelically knocked out mice.
(F) Comparison of the single- and triple-target CRISPR methods. The table shows the gRNA injection condition and resulting phenotypes (Figure S1G). Embryos,
number of injected and transferred embryos; Pups, number of pups born (total); Albino, pups with albino coat color (biallelic knockout); Pigmented, pups withmosaic or wild-type coat color.

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Tyr exon1
Chr7 (-)

Tyr exon5

87,493,000 bp

87,492,500 bp

87,429,500 bp

Target-1

87,429,000 bp

Target-2

Wild-type 100

50

Tyr #1 100

50

Target-3

Inter-exon deletion
and inversion
Intra-exon deletion and inversion

Tyr #2

Short deletion

50

Intra-exon deletion
and inversion

50

Intra-exon deletion
and inversion

Short deletion

Short insertion(legend on next page)

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triple-target CRISPR method could produce biallelic KO micewith >90% efficiency in a single generation. Furthermore, undetectable expression of TYR protein was confirmed by mass
spectrometry (MS) comparing with another independently produced triple-target CRISPR KO strain. TYR protein was notdetectable in two Tyr KO mice, whereas it was detected in one
Nr3a KO mouse (Figure S1I). Importantly, since a triple-target
CRISPR method achieves high efficiency (>90%) for the biallelic
KO of a single gene, it has a potential to achieve efficiency higher
than 80% even for the biallelic KO of dual genes, which is sufficient for reliable analysis of behavioral phenotypes.
The SSS Enables Non-invasive, Fully Automated,
High-Performance Sleep/Wake Phenotyping
To identify system-level molecular circuits in organisms, boththe efficient production of KO mice and simple but accurate
phenotyping are necessary. The standard method for phenotyping mammals’ sleep/wake states is to record EEG/EMG
data. Each short-period segment of EEG/EMG data, called an‘‘epoch,’’ is then annotated into one of three sleep/wake states(‘‘REM [rapid eye movement] sleep,’’ ‘‘NREM [non-REM] sleep,’’
and ‘‘awake’’), determined by the EEG/EMG pattern (Figure S2A).
Here, to obtain a non-invasive and fully automated method forsimple and accurate sleep/wake phenotype analysis, we developed the SSS (Figure 3A). For this method, we first focused on
the non-invasive phenotyping of sleep/wake states by recording
the respiration of an animal, since the different states exhibit
unique respiration patterns (Figure S2A).
Among the current methods for non-invasively recording the
respiration of mice (Glaab et al., 2007; Gordon and Ali, 1984),
whole-body plethysmography (WBP) is one of the most popularand promising. The correlation between respiration patterns and
EEG/EMG-based sleep/wake states was previously analyzed(Hernandez et al., 2012). However, previous studies mainly
examined short recording periods (less than 1 day) during
different states of consciousness, so the volume of the WBPchamber could be relatively small. Although a smaller chamber
size increases the sensitivity of the respiration recording, too
small a chamber decreases the animal’s comfort and can affectsleep/wake behavior. Therefore, no system has been developed
for recording for a week or more, which is critical for accurate
sleep/wake phenotyping. Increasing the size of the chamber requires a compensatory increase in the signal/noise ratio of the
WBP system itself.
To optimize the WBP system, we modeled it as an equivalent
electrical circuit (Figure 3B; Supplemental Experimental Procedures for the detailed equations)—essentially a band-pass filter
for a certain frequency of pressure change. By computer simulation, we optimized the resistance and capacitance in the WBPsystem to band-pass the 1- to 10-Hz flow (Figure 3C), which isthe physiological range for mouse respiration frequency. Basedon this analysis, we constructed a high-performance WBP sys-tem (Figure 3A), which exhibited a high signal/noise ratio forthe 1- to 10-Hz flow and allowed us to record detailed, longterm respiration patterns of mice non-invasively.
For simple and accurate sleep/wake phenotype analyses, fully
automated annotation of the recorded data is also critical. We
previously developed FASTER (an unsupervised fully automatedsleep staging method for mice based on EEG/EMG recordings)(Sunagawa et al., 2013), which avoids the subjective manual
annotation of EEG/EMG data. We applied the FASTER method
to the recorded respiration data to create a fully automatedannotation pipeline (Figure S2B; Supplemental Experimental
Procedures). Each epoch is annotated as either ‘‘sleep’’ or‘‘awake’’ fully automatically. Since REM state represented only
5% of the total epochs, which agrees with past documentation(Chemelli et al., 1999), we focused on a two-state classification
between sleep and awake states as the first-order approximation in this study. Simultaneous recordings of the respiration
flow and EEG/EMG enabled us to estimate the performance of
the SSS system (Figure 3D), resulting in the high accuracy of
95.3 ± 0.4% (n = 3) for the respiration-based sleep/wake phenotyping (Figures 3E and S2C). Importantly, the non-invasivenessof the recording and fully automated data analysis minimized
the variability and subjectivity of the procedures, thereby making
the sleep/wake phenotyping highly reproducible, as indicatedby the overlapping probability densities of sleep/wake statesamong three individuals (Figure 3E). We also note that even‘‘quiet awake,’’ which was defined in this study as the awake
state accompanied by the lower 33.3% of EMG values, wasannotated accurately (95.3 ± 1.3%; n = 3) by the SSS system(Figure S2D). Therefore, the SSS provides a non-invasive, fully
automated method for sleep/wake phenotyping.
High-performance in the SSS system enabled us to obtainvarious sleep/wake phenotypes based not only on daily sleep
time (or awake time) but also on the transition probabilities between sleep and awake states (s and w, respectively, in Pws
and Psw; Figure 3F) and on the sleep/wake amplitude, which isrelated to the sleep-time variation within the daily sleep cycle(Figure 3G; Supplemental Experimental Procedures). We notethat Pws and Psw were proposed to represent sleep drive andwake drive in a previous study (De Valck and Cluydts, 2003)
and that they are informative sleep/wake phenotypes. We also
defined the sleep/wake amplitude as the coefficient of variation(CV, the SD divided by the mean) of sleep time for each 10-min
bin for 24 hr (Figure 3G).
To evaluate the performance of the SSS system, we recorded
108 individual C57BL/6N male mice (6 weeks old) in the SSS
chamber for a week (Figure 3H; Table S1). To avoid the firstday effect, the recorded data from the second day to the seventh
day were analyzed by the SSS annotation pipeline. The results
revealed a daily sleep time of 735.7 ± 50.8 min (mean ± SD;
n = 108), which corresponded to a CV of 6.9%. The Pws and
Psw were 0.0379 ± 0.0068 and 0.0363 ± 0.0067, respectively,

Figure 2. Exome Analysis of Whole-Body Biallelic Tyr KO Mice Produced by Triple-Target CRISPR Method
Genome alignments of exome sequence reads. The genomic regions targeted by the three gRNAs were shown for wild-type, Tyr KO #1, and Tyr KO #2. The upper
part of each panel shows read coverage, and the lower part shows read alignments (rectangles). Horizontal lines between rectangles indicate read pairing.
Different types of mutations occurring at the target sites are highlighted as follows: inter-exon deletion and inversion (red rectangles); intra-exon deletion andinversion mutation (green rectangles); short deletion (red dots); and short insertion (black dots).

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A

B

C
I1

C2

I2

I

G1

G2

Gain (Pa·s/ml)

101

R2

R1

C1

C2

10-3.510-4.010-4.510-5.0100
10-1
10 0.5R1

10 0.0

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10-2 10-1 100 101 102
Frequency (Hz)

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24 0

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Time (hr)612 18Time (hr)

24 0

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Time (hr)F

Sens. 94.2 ± 1.4%
Spec. 97.1 ± 1.0%

−5
−10

Sleep

1 23

Wake

Sens. 97.6 ± 1.0%
Spec. 93.7 ± 1.5%

−20

24

0
PC1

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0.10

Sleep time

0.05

Sleep
Wake
Sleep transition rate
Pws

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Count

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Psw
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C57BL/6N, male (n = 108)

100%

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10

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Accuracy 95.3 ± 0.4%
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10

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24

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Amplitude

0.025

0.075
Psw

Figure 3. The SSS Enables Non-invasive, Fully Automated, High-Performance Sleep/Wake Phenotyping(A) The SSS system. Upper left: one mouse is housed in an SSS chamber. The chamber is connected to a water outlet, and food is stored in its ceiling, whichallows 14 days of continuous recording without additional handling of the chamber. Upper right: up to 36 SSS chambers are set in an SSS rack. The rack is(legend continued on next page)

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and the sleep/wake amplitude was 0.4462 ± 0.0530. To evaluate
the SSS system for other inbred strains and in females, we recorded C57BL/6J male mice, C57BL/6N female mice, andC57BL/6J female mice (n = 178, 72, and 173, respectively; Figure S2E; Table S1). Their tight distributions of sleep time showedthat the SSS provides a simple and high-performance method
for sleep/wake phenotyping.
To further validate the SSS, we recorded various known
sleep/wake phenotypes in mice. First, we tested mice whose
sleep/wake behavior was perturbed pharmacologically. Methamphetamine (MAP), a potent psychostimulant that promotes
wakefulness (Edgar and Seidel, 1997), was injected intraperitoneally (i.p.) into mice during the day, at zeitgeber time 2 (ZT2;
Figure S3A). The administration of 3 mg/kg MAP significantly
decreased sleep time and Pws (Figures S3B and S3C). These
results indicated that the decreased sleep time was due
to increased stabilization of the awake state rather than bydecreased stabilization of the sleep state.
To test the SSS system for the opposite phenotype (sleep-timeincrease), we injected i.p. a sleep-inducing drug, diazepam(DZP) (Radulovacki et al., 1984), at nighttime, ZT13 (Figure S3D).
Indeed, the administration of 5 mg/kg DZP significantly
increased the sleep time and Pws (Figures S3E and S3F), whichis the exact opposite phenotype observed in MAP injection. To
further confirm observed sleep-phenotype changes, we performed the drug administration experiment under simultaneousrecording of SSS and EEG/EMG (Figures S3H and S3I). The accuracy, sleep sensitivity, sleep specificity, wake sensitivity, andwake specificity of SSS against EEG/EMG-based sleep staging
were as high as those for wild-type animals (Figures 3D and 3E).
These results clearly show that SSS is highly sensitive to sleep/
wake pattern, even in animals under drug administration.
Furthermore, to test the SSS system’s ability to detect thesleep/wake phenotypes of genetically modified animals, we recorded the behavior of circadian mutants that lose their circadianrhythm under constant darkness (DD) (Laposky et al., 2005; Shiromani et al., 2004; Wisor et al., 2002). Three KO strains, Bmal1
KO (Shimba et al., 2011), Cry1/Cry2 (van der Horst et al.,1999), and Per1/Per2 double-KO mice (Bae et al., 2001) were re-corded for a week under LD conditions (light on at ZT0, light off at
ZT12; Figure S3J). All strains exhibited less sleep during the lightphase (an inactive phase of wild-type mice) and more sleep during the dark phase (an active phase for wild-type mice; Figure S3K). Both Pws during the light phase and Psw during thedark phase are decreased in Bmal1 KO and Cry1/Cry2 doubleKOs (Figures S3L and S3M). Although Per1/Per2 double-KOmice did not exhibit a significant decrease of sleep/wake amplitude, their daily sleep/wake patterns differed from those of wildtype mice (Figure S3K). In all strains, the total sleep time was not
significantly different from that of the C57BL/6N mice (Figure S3M; Table S1). Overall, these results also indicate that the
SSS can sensitively detect various sleep/wake phenotypes,
including the total sleep/wake time, transition probabilities between sleep and awake states, and sleep/wake amplitude.
Combined Use of a Triple-Target CRISPR
and SSS Enables Sleep/Wake Phenotyping, Even in
Double-KO MiceTo test the feasibility of combining efficient KO mouse production with automated sleep/wake phenotyping, we analyzed
sleep/wake phenotypes in different KO mouse lines producedby the triple-target CRISPR method described earlier. First, we
designed three gRNAs against the Bmal1 gene (Figure S4A).
Sleep/wake phenotyping was performed for animals with the
KO genotype, which was confirmed by qPCR and/or genomic
sequencing (Figures S4D and S4E; Tables S1 and S2). As expected, the generated Bmal1 KO mice showed a significantly
decreased sleep/wake amplitude under LD conditions compared with wild-type mice, and the Tyr KO mice did not (Figures
4A–4C; Figure S4H). In contrast, the daily sleep times did not
differ between the Tyr KO and Bmal1 KO mice or in comparison
with wild-type mice (Figure 4C). Notably, the Tyr KO mice
had sleep/wake patterns similar to those of the wild-typemice (Figure 4B), indicating that the triple-target CRISPR method
itself had little effect on sleep/wake phenotypes. Next, we generated double-KO mice for Cry1/Cry2 and Per1/Per2, which are
well-known mouse clock genes. We designed three gRNAs
each for each gene (Figures S4B and S4C), and sleep/wakeequipped with continuous air circulation units, which provides fresh air to each chamber. Lower panel: we used six SSS racks for recording and thus could
determine the sleep/wake phenotypes of 216 mice per week.
(B) Equivalent electric circuit model of the SSS system. The SSS chamber (red dashed box) was modeled with a capacitor (C1) and resister (R1) in parallel, and thepressure sensor (blue dashed box) was modeled with the resister (R2) and capacitor (C2) in series (Supplemental Experimental Procedures).
(C) Calculation of the conductance over R2, which corresponds to the amplification factor of the pressure detected with the sensor and optimization of C2 and R1
to maximize the gain of the frequency band of the physiological respiration of mice (1 to 10 Hz, pink shaded area). Based on the simulation results, C2 was fixed to
6.12 3 10 5 ml/Pa, and R1 was fixed to 172 Pa , s/ml.
(D) SSS-based sleep/wake staging results compared with conventional EEG/EMG-based sleep/wake staging results. Data from three mice were used. Uppergraph shows the sleep time for every 30 min evaluated by SSS. Lower graph shows the accuracy based on conventional EEG/EMG-based staging, for each
30 min during recording.
(E) The performance of SSS. Sleep (red) and awake (blue) states of three individual mice are shown as probability densities on a two-dimesional principalcomponent map in the SSS annotation pipeline. Values represent the mean ± SD (n = 3) of the sensitivity and specificity of sleep (red) and awake (blue) states
when compared with EEG/EMG-based sleep/wake staging. The overall accuracy reached 95.3% ± 0.4% (mean ± SD, green).
(F) Illustration of sleep/wake transitions. Upper left: schematic view of the transition probabilities between sleep and awake states. Upper right: sleep time atdifferent values of Pws and Psw. When both Pws and Psw decrease (green circle), both sleep and awake episodes will stabilize. Note that when both Pws and Psw
increase (red diamond), the stability of each state is dramatically decreased (bar chart below) without changing the total sleep (or awake) time.
(G) Illustration of sleep/wake amplitude. In the chart, different amplitudes are distinguished by color, and the points represent sleep times at various times of day.
(H) Distribution of sleep/wake parameters of C57BL/6N male mice recorded in the SSS. The pink and sky blue dashed lines show the mean and the mean ± SD ofeach distribution, respectively.
See also Table S1 and Supplemental Experimental Procedures.

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Bmal1 (n = 6)

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E

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Time (Hour)phenotyping was performed for animals with the KO genotype(Figures S4F and S4G; Tables S1 and S2). Both strains showedsimilar phenotypes with conventionally generated KO mice (Figures 4A–4C; Table S1). All three circadian KO strains were also
recorded for a week under DD conditions and confirmed to
exhibit arrhythmic phenotype (Figures S4I and S4J).
To further validate the functionality of our strategy, we designed three gRNAs against the Hcrt gene (Figure S4K) and produced animals by triple-target CRISPR method. Sleep/wakephenotyping was performed for animals with KO genotype (Figure S4L; Tables S1 and S2). Consistent with the conventional
Hcrt KO mice (Chemelli et al., 1999), the mice showed significant
increase in both transition probabilities, Pws and Psw, without any
significant change in total sleep time (Figures 4D–4F and S4M).
Overall, the combinatorial use of triple-target CRISPR andSSS enables the efficient generation of KO mice and their accurate sleep/wake phenotyping not only in single-KO mice but alsoin double-KO mice.
Reverse Genetics without Crossing Reveals Nr3a
Mutant to Be a Short-Sleeper Mouse
Mammalian reverse genetics without crossing can be applied tothe comprehensive analysis of gene functions at the systems
and whole-organism level. To demonstrate proof of concept,

60
Sleep time (min)A

0Figure 4. Combined Use of a Triple-Target
CRISPR and SSS Enables Sleep/Wake Phenotyping in Single- and Double-KO Mice(A) Sleep time (per hour) phenotype for individual
mice in Tyr KO mice and circadian-clock mutants
produced by triple-target CRISPR.
(B) Sleep time (per hour) over 24 hr averaged over
6 days in Tyr KO mice and circadian-clock mutants
produced by triple-target CRISPR. Red lines show
the mean sleep time at each time of day for eachstrain. Gray line shows the data for C57BL/6N malemice (n = 108). The shaded area around the line is
the SEM for each time point.
(C) Distributions of sleep/wake parameters of Tyr
KO mice and circadian-clock mutants. Dunnett’s
test compared to C57BL/6N male mice.
(D) Sleep-time (per hour) phenotype of Hcrt KO
mice.
(E) Sleep time (per hour) over 24 hr averaged over
6 days in Hcrt KO mice. Red line, gray line, and theshaded area are the same as described in (B).
(F) Distributions of sleep/wake parameters of Hcrt
KO mice. Dunnett’s test compared to C57BL/6N
male mice. See also Table S1.

we comprehensively targeted the members of the NMDAR family. We focused
Amplitude
on this family because the NMDAR is
implicated in several psychological disorders, including schizophrenia (Belforte
et al., 2010; Moghaddam and Javitt,
0.02
0.04
0.06
P sw
2012; Mohn et al., 1999), Alzheimer’s disease (Snyder et al., 2005), and depression(Autry et al., 2011; Li et al., 2010), all ofwhich have associated sleep disorders.
There are seven genes in the NMDAR family: Nr1, Nr2a, Nr2b,
Nr2c, Nr2d, Nr3a, and Nr3b. For triple-target CRISPR, we
designed three gRNAs for each of these genes (Figures S5A–
S5G) and used them to generate KO mice for each one.
Sleep/wake phenotyping was performed for animals with the
KO genotype, which was confirmed by qPCR and/or genomic
sequencing (Figures S5H–S5L; Tables S1 and S2). First, we
confirmed that the lethal phenotype seen in Nr1 and Nr2b biallelic KO mice that was generated by conventional methods (Forrest et al., 1994; Li et al., 1994) also occurred in Nr1 and Nr2b KO
mice generated by the triple-target CRISPR method, because
we noted that there were no survivors of Nr1 and Nr2b KO
genotypes (Table S2). KO mice for the other five NMDAR genes,
Nr2a, Nr2c, Nr2d, Nr3a, and Nr3b, survived for at least 6 to
7 weeks (Table S2), at which time they were placed in SSS chambers, and their sleep/wake behavior was recorded for at least
1 week under LD conditions. For the phenotype analysis, weused the 6-day data from the second recording day to avoid
the first-day effect (Figure 5A). The sleep/wake phenotype analysis was performed fully automatically, and the statistical evaluation used sleep time, Pws, Psw, and sleep/wake amplitude asparameters (Figures 5B, 5C, and S5M; Table S1).
Among the five KO mice, the Nr3a KO mice showed a significant ‘‘short-sleeper’’ phenotype, with a daily sleep time of

8 Cell Reports 14, 1–16, January 26, 2016 ª2016 The Authors
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639.0 ± 17.5 min (n = 8), which is 96.7 min shorter than that ofwild-type mice (p < 0.0001). Nr3a KO mice displayed a lower
Pws than wild-type mice, whereas their Psw was normal (Figure 5C), indicating that their short-sleeper phenotype was due
to an increase in awake-state stabilization (i.e., longer episodedurations of the awake state) but not a decrease in sleep-statestabilization. To preclude the possibility of an off-target effectof CRISPR/Cas9 gene editing, we generated another group of
Nr3a KO mice (n = 12) by an independent CRISPR probe set(set 2; Figures S6A and S6B; Table S2) and confirmed theobserved short-sleeper phenotype compared with wild-typemice (p < 0.0001) (Figures 5D–5F and S6C). Furthermore, toensure that the short-sleeper phenotype is not due to the modality of sleep evaluation, we produced another series of Nr3a (set 1)
KO mice (Figure S6D) and used EEG and EMG recordings toevaluate their sleep. Consistent with the SSS results, EEG recordings showed a significant short-sleeper phenotype in KOmice (p < 0.05; Figure S6E). For genomic validation, four of thegenerated Nr3a KO mice (Figures S5K and S6B) mentioned
earlier were analyzed with exome sequencing (Table S5). In
both set 1 and set 2 animals, the analysis revealed deletions ofgenome stretches with variable lengths at the target site (Figure 6; Figure S7A), with its extent for at least one target site of
up to 100% (see Figure S7B for a magnifiable view). Importantly,
no off-target mutation was identified in this exome sequencing.
These results strongly suggested that the observed shortsleeper phenotype of Nr3a KO mice cannot be attributed to thepossible off-target effect of CRISPR but rather to the common
genomic defects in the Nr3a gene. Finally, to confirm theabsence of NR3A protein in the KO animals, we have performed
MS analysis in these animals (Figure S6F). Two Tyr KO animals(Figure S4D) were used as controls for NR3A protein detection,
which showed a clear peak at the position of internal standardof NR3A protein. By contrast, none of the Nr3a KO mice (Figures
S5K and S6B) had a detectable peak at the position of internal
NR3A protein. These results suggest that Nr3a is knocked
out not only at the genomic level but also at the protein expression level. Overall, these results demonstrate that the use of
mammalian reverse genetics without crossing, and specificallycombining this efficient KO mouse production with accurate,
automated phenotyping, enables the comprehensive identification of shared and diverse functions of gene family members inorganisms.
Developing a Database for the Triple-Target CRISPR
MethodAs described earlier, triple-target CRISPR enabled us to efficiently produce biallelic KO mice. However, as we produced
several KO mice using this method, we noticed that manuallydesigning triple targets for the same gene is still time consuming,
even using existing tools to extract candidate target sequences
for a given genomic sequence (http://cas9.cbi.pku.edu.cn/
index.jsp) (Ma et al., 2013) and to evaluate the off-target riskfor each candidate (http://tools.genome-engineering.org) (Ranet al., 2013). This manual design procedure takes up to a few
hours for a single gene and, therefore, limits the ability to performlarge-scale screenings. Therefore, we sought to develop an
automated method that performs all of the gRNA selection steps.

We scanned the whole mouse genome once, and all of the suitable targets are now in an online database (Figure 7A; Supplemental Experimental Procedures). This gRNA database provides
at least three target sequences (one set for triple-target CRISPR)
for 81.2% of mouse genes. In addition, 71.9% of all mouse genes
have more than six target sequences (multiple sets for tripletarget CRISPR), which are also included (Figures 7B and 7C).
To validate the usefulness of the triple-target CRISPR database, we selected two independent sets of triple targets for
the Tyr gene (Tyr-4, Tyr-5, and Tyr-6 for set 2, and Tyr-7, Tyr-8,
and Tyr-9 for set 3; Figure 7D), which were distinct from Tyr-1,
Tyr-2, and Tyr-3 (set 1) used in the previous experiments (Figure 1C). When injected into fertilized eggs, the triple-target
method using both sets of gRNAs showed almost perfect efficacy (96.6% and 100%, respectively; Figures 7E and 7F), supporting the usefulness of the triple-target CRISPR database.
Therefore, we made this database public (http://crispr.riken.jp/).
DISCUSSION
Triple-Target CRISPR Enables the Efficient Productionof Biallelic KO Mice in a Single Generation
In this study, we sought to achieve the highly efficient (>90%)
production of whole-body biallelic KO mice in a single generation.
We used a simple computational model to estimate the minimum
efficacy of different CRISPR methods and discovered that multiple-target CRISPR methods would generate mutant mice withmuch higher efficiency than single-target CRISPR. We designed
three different gRNAs against the Tyr gene and confirmed thatthe triple-target CRISPR method—using a mixture of three gRNAs(Tyr-1, Tyr-2, and Tyr-3)—achieved almost perfect efficacy(97.5%), whereas the single-target method with any of the gRNAs
at a 3-fold higher concentration had only moderate efficiency.
Genomic verification revealed no off-target mutation, at least inexons (Figure 2; Table S5). The total number of gigabase pairs(or Gbp) read in the exome sequencing including Tyr (set 1),
Nr3a (set 1), and Nr3a (set 2) KOs was 65.6 Gbp. This is morethan 24 times greater than the whole genome base pairs of mouse(2.7 Gbp). This, therefore, gives us enough coverage to be confident that off-target effect of the triple-target CRISPR strategy is
not a problem in practice. Importantly, the DNA-cleavage efficiency of Tyr-3 was more than 3-fold higher than that of Tyr-2 (Figure 1D), whereas the whole-body biallelic KO efficiency for Tyr-3(64.7%) was similar to that for Tyr-2 (54.2%) (Figure 1F), indicatingthat a limiting factor in vivo is not DNA cleavage by the CRISPR/
Cas9 system but other factors such as DNA repair, consistent
with the prediction by the computational model. The reproducibility and robustness of the triple-target method were further
confirmed by additional experiments with two independent setsof three gRNAs against the Tyr gene (Figures 7E and 7F). We
also confirmed the KO efficiency by genotyping all mice used
for SSS analysis in this study. We genotyped 102 mice producedby the triple-target CRISPR method, including 11 single-KO and 2
double-KO strains (Table S2). At least 92.2% (n = 94) of the animals were confirmed as KO mice by qPCR or sequencing. Thisefficacy is confirmed to be higher than the minimum efficacy predicted by our model. In addition, the triple-target CRISPR method
is scalable and can be applied to the identification of common andCell Reports 14, 1–16, January 26, 2016 ª2016 The Authors 9

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Nr2c (n = 15)

Nr2d (n = 13)

Nr3a (set 1) (n = 8)60

Nr3b (n = 11)

Mouse

Nr2a (n = 8)

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Sleep time (min)A

Time (Day)0

Sleep time (min)B
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Nr2c (n = 15)

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Nr3b (n = 11)

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700

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900

Sleep time (min)

0.2

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Time (Hour)
Figure 5. Reverse Genetics without Crossing Reveals Nr3a Mutant to Be a Short-Sleeper(A) Sleep-time (per hour) phenotype for all individual KO mice of five NMDA receptor family members generated by triple-target CRISPR.
(B) Sleep time (per hour) over 24 hr, averaged over six days in KO mice of five NMDA receptor family members. Red lines show the mean sleep time at each time of
day for each strain. Gray line shows the data for C57BL/6N male mice (n = 108). The shaded area around the line is the SEM for each time point.
(C) Distributions of sleep/wake parameters of comprehensive KO mice of NMDA receptor family members. Dunnett’s test or Dunnett’s modified Tukey-Kramer
pairwise multiple comparison test compared to C57BL/6N male mice.
(D) Sleep-time (per hour) phenotype of Nr3a KO mice (set 2) generated by triple-target CRISPR.
(E) Sleep time (per hour) over 24 hr, averaged over 6 days in Nr3a KO mice (set 2). Red line, gray line, and the shaded area are the same as described in (B).
(F) Distributions of sleep/wake parameters of Nr3a KO mice (set 2). Dunnett’s test or Dunnett’s modified Tukey-Kramer pairwise multiple comparison testcompared to C57BL/6N male mice.
See also Table S1.

diverse functions of a gene family of interest (Figure 5). Furthermore, the triple-target CRISPR method is applicable to the phenotype analysis of double-KO mice (Figures 4A–4C). We also

created a publicly available CRISPR database that provides triple-target candidate gRNAs for more than 81.2% of the genes
in the mouse genome (Figure 7A).

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The SSS Provides a Simple and Accurate Phenotyping
Method for a Complex Behavior
Along with the efficient production of biallelic KO mice, accurate
phenotype analysis is also important to make full use of mammalian reverse genetics without crossing. In this study, we focusedon the phenotype analysis of sleep/wake states, because sleep
disorders are often associated with psychiatric diseases. The
development of a non-invasive and fully automated method forsleep/wake phenotyping (the SSS) allowed us to sensitively
detect the sleep-time effects of drugs targeting the CNS,
including a psychostimulant, MAP (dopamine transporter antagonist), and a known sleep-inducing drug, DZP (GABAA receptor
agonist) (Figures S3A–S3F). In addition, the SSS enabled us toaccurately and quantitatively analyze other sleep/wake phenotypes, such as sleep/wake amplitude, which was shown by thedecreased amplitude of clock mutants (Figure 4C), as well as
the transition probabilities from sleep to awake states, andfrom awake to sleep states (Figures 3F–3H), which was clearlyshown by the significant increase of Pws and Psw in Hcrt KO
mice (Figure 4F). This ability is useful for dissecting the effectsof CNS drugs on daily sleep time. For example, we found thatthe decreased sleep time caused by MAP was due to an increase
in awake-state stabilization (low Pws) rather than to a decrease in
sleep-state stabilization (Figure S3B). In contrast, the increased
sleep time caused by DZP for 5 hr after the injection was due to adecrease in awake-state stabilization (high Pws) (Figure S3E),
suggesting that the sleep/wake phenotype induced by DZP is
functionally the opposite of that induced by MAP. Interestingly,
after DZP increased the sleep time (0 to 5 hr after injection), it
conversely decreased the sleep time (5 to 10 hr after injection)(Figure S3G). This compensatory decrease in sleep time by
DZP was associated with a slight decrease in sleep-state stabilization (high Psw) (Figure S3G; p = 0.0869). These results suggestthat the SSS can sensitively detect and carefully dissect
the common and diverse sleep/wake effects of drugs targeting
the CNS.
The sleep time of two circadian KO animals, Bmal1 KO and
Cry1/Cry2 double-KO mice, recorded in the SSS is not exactly
the same as in past reports (Laposky et al., 2005; Wisor et al.,2002). This might be partly because of the functional advantages of the SSS system. First, because of its convenience,
the number of recorded mutant mice is larger in this study,
where 34 Bmal1 KO and 27 Cry1/Cry2 KO mice and morethan 100 wild-type mice are recorded. In contrast, nine
Bmal1 KO mice against eight wild-type mice are recorded in
the Laposky et al. (2005) paper, and six Cry1/2 double-KOmice against eight wild-type mice are recorded in the Wisor
et al. (2002) paper. Second, in both studies, the sleep phenotypes were evaluated by electrodes implanted in the skull,
which is inevitably invasive. In fact, three out of nine KO mice
were removed from the study after EEG/EMG recording in the
Laposky et al. paper because EEG/EMG signals were deterio-

rating, which implies some invasiveness inherited in the conventional EEG/EMG recording methods.
Several other approaches have been used in the past to
develop automated non-invasive sleep staging systems. The
video-based sleep stager is non-invasive and can discriminate
between REM and NREM (McShane et al., 2012). However, theexpected drawback is difficulty in parallelization due to thehuge amount of data to be handled. Furthermore, this method
requires a user to set a parameter, which makes it distinct
from the SSS, which requires no manual optimization. Piezoelectric-based movement detection technology allowed for estimation of the sleep/wake status from the animal motion (Flores
et al., 2007). The simplicity of this system is attractive, althoughwhen performing a high-throughput phenotyping in this system,
we expect its inevitable interference, i.e., the difficulty in assigning the movement information to the recoding animal against theinevitable noise coming from the neighboring chambers. In
contrast, the SSS only uses single-channel respiration flow information per animal, which is a very compact-sized dataset andthe respiration is detected only by the pressure inside the chamber, which is much less prone to be affected from the neighboring chambers. These characteristics make the SSS a much
useful technology.
Regulation of Sleep/Wake States by NMDAR Family
Members
To test the use of our combined method to comprehensively
identify the common and diverse functions of gene family members, we separately knocked out all of the NMDAR family members. We found that Nr3a KO mice are short-sleeper mutants.
Nr3a expression peaks during early postnatal life and decreases
to lower levels in adulthood (Henson et al., 2010). Unlike thetraditional NMDARs, Nr3a-containing NMDARs have less Ca2+
permeability and are almost completely insensitive to Mg2+ blockat hyperpolarized potentials (Sasaki et al., 2002; Tong et al.,2008). Recently, adult Nr3a KO mice were reported to haveincreased pain sensation and enhanced abilities in learningand memory tasks (Mohamad et al., 2013). These KO mice hadgreater long-term potentiation (LTP) in adult hippocampal slices
compared with wild-type slices. Based on these previous findings, we can assume two possible hypotheses to explain the
short-sleeper phenotype in Nr3a KO animals. One is the developmental circuit influence of Nr3a-containing neurons. Since
Nr3a defect impairs fundamental NMDAR responses that are
deeply related to formation of synaptic networks, the absenceof Nr3a may alter the original sleep regulation network of thebrain. The other is the direct neural activity modulation by
NR3A-containing neurons. Even Nr3a impairment during development may be complemented by another mechanism; the influence of Nr3a modulation against neural activity remains until
adulthood. The enhanced LTP can be explained by decreased
blunting of the NMDAR response by Nr3a. Because the NMDAR

Figure 6. Exome Analysis of Whole-Body Biallelic Nr3a, Set 1, KO Mice Produced by Triple-Target CRISPR Method
The genomic regions targeted by the three gRNAs were shown for two mice in Nr3a (set 1) KO group (Figure S5K). The upper part of each panel shows readcoverage, and the lower part shows read alignments (rectangles). Horizontal lines between rectangles indicate read pairing. Different types of mutations
occurring at the target sites are highlighted as follows: inter-exon deletion (red rectangles); intra-exon deletion mutation (green rectangles); short deletion (reddots); and short insertion (blue dots).

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AMatch #
in genome
GCAGCTGGGGACACAGCAGCTGG >1GCAGGTGATGAAGCCACAGGATTCTGG >1GTGA
GTTTTTCTATGCAGGTAAGAAGG
GTTT1GTGCGCTACGCGTGCGCGGTGGG
GTGC
1GCAAAGACCTCATGGGCTTCGGG
GCAA
1GAGCAGATTGTGGTGGCTCTCGG
GAGC
1GATGGGTAACTGAGAAATACGGG
GATG
1
GAGGTATATTGATCTGCTTCAGG
GAGG
1Reverse primer
AAAAGCACCGACTCGGTGCCAAAAAAGCACCGACTCGG-TGCC
AAA
A
GCAAAG-ACCTCATGGGCTTCGGGGCAAAGACCTCATGGGCTTCGGG

GATGGGTAACTGAGAAATACGGG
AACTGAGAAATGAGCAGATTGTGGTGGCTCTCGG
GAG
GATGGGTAACTGAGAAATACGGGGAT
GAGGTATATTGATCTGCTTCAGG
GAG

Multiple match removal

TargetPrimer Homologs RemovalPri

Off-target

Off-target Filtering
GAGGTATATTGATCTGCTTCAGG
ATTGATC
ATCTGCT

Pattern based extraction

Low AT, TTTT Removal

PatternG
AnnnnnnnnnnnnnnnnnnnnGG
C
GCAGCTGGGGACACAGCAGCTGG
GTGATGAAGCCACAGGATTCTGG
GTGA
GTTTTTCTATGCAGGTAAGAAGGGTTTGTGCGCTACGCGTGCGCGGTGGGGTGC
GCAAAGACCTCATGGGCTTCGGG
G
CAA
GAGCAGATTGTGGTGGCTCTCGG
G
AGC
GATGGGTAACTGAGAAATACGGG
G
ATG
GAGGTATATTGATCTGCTTCAGG
GAGG

Structure based Filtering

GTTTTTCTATGCAGGTAAGAAGG
GTTTGTGCGCTACGCGTGCGCGGTGGG
GT
TGC
U
U
G

GCAAAGACCTCATGGGCTTCGGG
GCAA
GAGCAGATTGTGGTGGCTCTCGG
GAGC
GATGGGTAACTGAGAAATACGGG
GATG
GAGGTATATTGATCTGCTTCAGG
GAGG

A

CU

A U

U G
C
G G A
G C
U C
G A
U U UC C
G
U
G
U
G
A
G
A
U C
A
U U
G G A A UU G A A
C
A A
A U U
U A

C
U G
U
U
G AA GUC U AGU AUUU AA
A G AU AUCGGG A U G CA A
A
UA
A A

G
C
C

U G
C A
G G
G C
U C
G A
U U UCGCUGU G A A AA
A U G
GU AU
A C U U A
G
U
G
A
G
A
C
C
C
GU
G
C C CGUU AU
G
G CU A GU U
C G A AG
U
U AU
U
U AA
A
A G UAUGC C AGAU
G A AGUA
A A

Database StorageGATGGGTAACTGAGAAATACGGGGTAACTGAGAAGAGCAGATTGTGGTGGCTCTCGG
GTGGCTCTCGG GAGGTATATTGATCTGCTTCAGG
ATATTGATCTG

B

C
Number of Genes
15000

20000

25000

2500
2000

14

>14

12−

6−8

9.3%

1
9−1

3−5

18.8%

10000

1500

Count

5000

0−2

Number ofTargets

0

71.9%

1000
500

Less than
two targets

A single set of
triple target

Multiple sets of triple targets

0
0

25
50
75
Number of targets

100

D
Tyr target sequence-4
ggacctcagttccccttcaa (+)

chr7 (-)

Tyr target sequence-5
agtttgacccagtatgaatc (+)

87,490,000

Tyr target sequence-6
gagtctctgttatggccgat (-)

87,460,000

Set 2

87,430,000
Tyrggtcatccacccctttgaag (-)
Tyr target sequence-7

E

Triple target
Set 2 (Tyr-4,5,6)

aagccatggccagatacgac (-)
Tyr target sequence-9

gtgttgacccattgttcatt (-)
Tyr target sequence-8

Triple target
Set 3 (Tyr-7,8,9)

Set 3

F

Triple Set 2(Tyr-4,5,6)

Triple Set 3(Tyr-7,8,9)

gRNA1(ng/µl)

50

50

gRNA2
(ng/µl)

50

50

gRNA3
(ng/µl)50

50

Embryos

145

105

Pups

32

14

Albino31 (96.9%)

14 (100%)

Pigmented

1 (3.1%)0 (0%)(legend on next page)

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Please cite this article in press as: Sunagawa et al., Mammalian Reverse Genetics without Crossing Reveals Nr3a as a Short-Sleeper Gene, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2015.12.052

family can be strongly blocked by MK-801, which results in hyperlocomotion when administered to mice (Koek et al., 1988),
it is possible that, without Nr3a, the NMDAR response is weakened, and this may result in shorter sleep. To test these hypotheses, conditional perturbation of Nr3a-containing neurons isnecessary in future investigations.
Although the detailed molecular mechanism, in which Nr3acontributes to the sleep/wake cycle, remains to be elucidated,the discovery of Nr3a KO mice as a strong short sleeper provides
an interesting entry point for mammalian sleep research. We also
note that, in the fruit fly, forward genetic screenings have shownthat other ion channels play important roles in sleep homeostasis. Mutation in the Shaker gene, which encodes a voltagedependent potassium channel, and in the Redeye gene, which
encodes a nicotinic acetylcholine receptor a subunit, both resultin a short-sleeping phenotype (Cirelli et al., 2005; Shi et al., 2014).
Therefore, it will be interesting to investigate the relationship ofthe mammalian homologs of these channels to NR3A-containing
NMDARs in mammalian sleep.
Toward Organism-Level Systems Biology
In this study, we sought to develop mammalian reverse genetics
without crossing by eliciting the efficient (>90%) production andaccurate (>90%) phenotype analysis of mutant animals in a single generation. Mammalian reverse genetics without crossing(called ‘‘next-generation’’ mammalian reverse genetics) now allows us to test—in whole organisms—hypotheses that arederived from the past knowledge. For example, a computationalmodel of slow-wave sleep that provides explanations for theobserved complex dynamics of sleep/wake states, along withpredictions for the important molecular pathways regulatingthese states, could be extremely powerful. Previously, it wasdifficult to test such predictions, at least without years of labor.
However, the present study demonstrated that the importanceof a predicted molecular pathway can be efficiently tested using
next-generation reverse genetics. Therefore, one of the nextchallenges will be to construct a simple computational modeland experimentally validate its predictions using triple-target
CRISPR and/or the SSS. New research fields, including the system-level identification and analysis of molecular circuits inwhole organisms, lie ahead.

12-hr/12-hr light/dark cycle. All experimental procedures and housing conditions involving animals and their care were in accordance with the RIKEN Regulations for Animal Experiments and the Animal Care and the Use Committeeof the Graduate School of Medicine, The University of Tokyo.
One-Cell Embryo Microinjection
C57BL/6N females (4–6 weeks old) were superovulated and mated with
C57BL/6N males. Fertilized eggs were collected from the ampulla of theoviduct of plugged C57BL/6N females by micro-dissection and kept in
KSOM medium (Merck Millipore) in a 5% CO2 incubator at 37 C. Cas9mRNA (100 ng/ml) and gRNAs (150 ng/ml in total) were co-injected into the cytoplasm of fertilized eggs in M2 medium (Merck Millipore) at room temperature.
Details of the cytoplasmic injection were described previously (Sumiyama
et al., 2010). After microinjection, the injected embryos were cultured for 1 hr
in KSOM medium (Merck Millipore) in a 5% CO2 incubator at 37 C, and
15–30 embryos were then transferred to the oviducts of pseudopregnant
ICR female mice.
Non-invasive Sleep/Wake Phenotype Analysis by SSS Recordings
Mice were placed in the SSS chamber (Figure 3A, upper left panel; Minamide
Rika Shokai), which had an air flow of at least 0.4 l/min. The chamber was connected to the SSS sensor (PMD-8203-2G, Biotex, light brown box on top of thechamber in Figure 3A), which detected the pressure difference between theoutside and inside of the chamber. The analog signal output from the sensor
was digitized at 250 Hz by an AD converter (ADS-0128aK-64MB, Microscience) and stored on a computer by data logging software (Lab-DAQ Pro,
Matsuyama Advance). The analysis of the digitized data was fully automatedas follows: first, the respiration time domain data were converted to frequency
domain data by fast Fourier transformation. The power spectrum was thensubjected to principal-component analysis. The first three principal components were used for clustering by probability density clustering (Azzalini andTorelli, 2007). Finally, the clusters were annotated according to the power
spectrum of each cluster. The details in each step of SSS annotation pipeline
are described in the Supplemental Experimental Procedures. One set of data
recording in the SSS chamber was for 2 weeks. During the experiment, neither
the chamber nor the rack was opened unless the injection was performed i.p.
The light condition of the SSS rack was set to a 12-hr/12-hr light/dark cycle,
and food and water were given ad libitum. Data analysis was conducted
6 days from the second recording date.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, five tables, and a data file showing the source code of the
SSS analysis and can be found with this article online at http://dx.doi.org/10.
1016/j.celrep.2015.12.052.
AUTHOR CONTRIBUTIONSEXPERIMENTAL PROCEDURES
Animals
C57BL/6NJcl mice were purchased from CLEA Japan, and C57BL/6J mice
were purchased from Oriental Yeast. All mice were given food and water ad
libitum. They were kept in an environment at an ambient temperatureof 21 C, with a relative humidity of 50%. The light was controlled under a

H.R.U. and G.A.S. designed the study. G.A.S. developed the SSS system andperformed the sleep data analysis. G.A.S. and S.S. performed the SSS
recording. G.A.S., D.T., and K.L.O. performed EEG/EMG recordings. K.S.
developed the triple-target CRISPR method. M.U.-T. designed the CRISPR
probes. M.U.-T. and S.S. produced CRISPR probes. K.S. and R.O. produced
the mice. D.P. implemented the whole-genome detection method and the

Figure 7. A Publicly Available Database for the Triple-Target CRISPR Method(A) The pipeline for automatically selecting targets for the triple-target CRISPR method.
(B) Distribution of the number of the targets per gene.
(C) Histogram of the number of targets per gene.
(D) Alternative target sequences for the Tyr gene.
(E) Experimental validation of the automatically selected targets for the Tyr gene. Two sets of additional triple gRNAs (Tyr-4, Tyr-5, and Tyr-6 for set 2 and Tyr-7,
Tyr-8, and Tyr-9 for set 3) were tested. Photographs show the coat color of the generated mice.
(F) Table shows the injection conditions of the gRNAs and resulting phenotype. Embryos, number of injected and transferred embryos; Pups, number of born
pups (total); Albino, pups with albino coat color (biallelic knockout); Pigmented, pups with mosaic or wild-type coat color.

14 Cell Reports 14, 1–16, January 26, 2016 ª2016 The Authors
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Please cite this article in press as: Sunagawa et al., Mammalian Reverse Genetics without Crossing Reveals Nr3a as a Short-Sleeper Gene, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2015.12.052

database for the triple-target CRISPR method. H.U. developed the SSA assay.
M.U.-T. and H.F. performed the genotyping of animals. O.N. and S.K. performed the exome sequencing analysis. R.N. and Y.S. performed the protein
quantification. H.R.U. and G.A.S. wrote the manuscript. All authors discussed
the results and commented on the manuscript text.
ACKNOWLEDGMENTS
We thank T. Mikami for the enthusiastic support for developing the SSS. We
also thank all the lab members at RIKEN CDB, QBiC, and The University ofTokyo, in particular, S. Fujino, J. Hara, J. Yoshida-Garcon, M. Shiokawa, A.
Kishimoto, A. Nishiyama, A. Sato, Y. Wada, and M. Nomura for their kind
help in preparing the materials and supporting experiments; the LARGE,
RIKEN CDB for housing the mice; Kaori Tatsumi in the Phyloinformatics Unit
(GRAS), RIKEN Center for Life Science Technologies, for exome library preparation and sequencing; T. Ueno for running the initial experiment for tripletarget CRISPR; F. Tatsuki and G. Kanda for helpful discussion; and K. Yoshida
for improving the SSS algorithm. This work was supported by the Innovative
Cell Biology by Innovative Technology Program from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of Japan (H.R.U.); a Grant-inAid for Scientific Research (S) (grant no. 25221004; H.R.U.) and a Grant-in-Aid
for Scientific Research on Innovative Areas (grant no. 23115006) from MEXT/
Japan Society for the Promotion of Science (JSPS; H.R.U.); a Grant-in-Aid forYoung Scientists (B) (grant no. 26870858; G.A.S.); a Grant-in-Aid for Scientific
Research on Innovative Areas (grant no. 26113720; K.S.); the strategic
programs for R&D (President’s discretionary fund) of RIKEN (H.R.U.); an
intramural Grant-in-Aid from the RIKEN Center for Developmental Biology
and RIKEN Quantitative Biology Center (H.R.U.); a grant from AMED Core
Research for Evolutionary Medical Science and Technology (AMED-CREST),Japan AMED (H.R.U.), and Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST; H.R.U.); theProgram for the Brain Mapping by Integrated Neurotechnologies for Disease
Studies (Brain/MINDS) from the MEXT (H.R.U.); the Basic Science and Platform Technology Program for Innovative Biological Medicine from the Japan
Agency for Medical Research and Development (H.R.U.); and by the RIKEN
Foreign Postdoctoral Researcher Program (D.P.).

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