1075002

review-article2022

JBRXXX10.1177/07487304221075002Journal of Biological RhythmsMinami et al. / SHORT TITLECIRCADIAN MEDICINEHigh-throughput Genetically Modified
Animal Experiments Achieved by Next-generation
Mammalian Genetics
Yoichi Minami*, Yufei Yuan*, and Hiroki R. Ueda*,†,1*
Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japanand †Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, Suita, Japan
Abstract Animal models are essential tools for modern scientists to conduct
biological experiments and investigate their hypotheses in vivo. However, forthe past decade, raising the throughput of such animal experiments has beena great challenge. Conventionally, in vivo high-throughput assay was achieved
through large-scale mutagen-driven forward genetic screening, which took
years to find causal genes. In contrast, reverse genetics accelerated the causal
gene identification process, but its throughput was also limited by 2 barriers,that is, the genome modification step and the time-consuming crossing step.
Defined as genetics without crossing, next-generation genetics is able to produce gene-modified animals that can be analyzed at the founder generation(F0). This method is or can be accomplished through recent technologicaladvances in gene editing and virus-based efficient gene modifications.
Notably, next-generation genetics has accelerated the process of cross-species
studies, and it will be a useful technique during animal experiments as it can
provide genetic perturbation at an individual level without crossing. In thisreview, we begin by introducing the history of animal-based high-throughput
analysis, with a specific focus on chronobiology. We then describe ways thatgene modification efficiency during animal experiments was enhanced andwhy crossing remained a barrier to reaching higher efficiency. Moreover, we
mention the Triple CRISPR as a critical technique for achieving next-generation genetics. Finally, we discuss the potential applications and limitations of
next-generation mammalian genetics.
Keywords triple CRISPR, ES mouse, next-generation genetics, circadian rhythms, highthroughput assay

Animal models have contributed to the elucidation of mechanisms in various life systems.
Specifically, studies using non-mammalian organisms, such as Escherichia coli, Caenorhabditis elegans,
Saccharomyces cerevisiae, Schizosaccharomyces pombe,

Neurospora, Cyanobacteria, and Drosophila melanogaster, have considerably contributed to the development of genetics and molecular biology. Moreover,studies on the KaiA-C in cyanobacteria and the clock
genes in Neurospora crassa, such as FRQ, have made1. To whom all correspondence should be addressed: Hiroki R. Ueda, Department of Systems Pharmacology, Graduate
School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan; e-mail: uedah-tky@umin.ac.jp.
JOURNAL OF BIOLOGICAL RHYTHMS, Vol. XX No. X, Month 202X 1­–17https://doi.org/10.1177/07487304221075002
DOI:
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important contributions to the molecular mechanisms of the biological clock (Dunlap, 1999; Dunlap
and Loros, 2004; Iwasaki and Kondo, 2004; Cohen
and Golden, 2015; Dunlap and Loros, 2017; Loros,2020). Epoch-making studies have utilized fruit
flies in chronobiology, that is, isolation of per0, pers,
and perl mutant flies, identifying causal gene period
and proposing the circadian oscillation circuit basedon the transcriptional-translational feedback loop
(Konopka and Benzer, 1971; Reddy et al., 1984;
Zehring et al., 1984; Hardin et al., 1990).
Mammalian studies have also contributed to life
science, especially in the biomedical field. Rodents,
such as hamsters, guinea pigs, rats, and mice, havebeen involved in animal experiments. Among these,mice are considered the king of model animals
because (1) they are easy to handle (small storage
space and easy to breed); (2) they have relatively
short life cycles; (3) many inbred lines have been
established; and (4) it is possible to generate genemodified mice such as transgenic (TG), knockout(KO), and knock-in (KI) mice. Conventionally, thethroughput of experiments involving geneticallymodified animals was relatively low, but the situation changed due to recent technological advances. In
this review, we focused on high-throughput assays
using mice supported by biotechnological advances.
In addition, we summarized the history of highthroughput assays in chronobiology, discussed thebarriers for raising throughputs, and introduced the“next-generation mammalian genetics,” which isdefined as genetics without crossing (Susaki et al.,2017; Ukai et al., 2017, 2019). Moreover, applicabletargets for next-generation genetics, such as genetically humanized animals, were also discussed.

ENU Mutant Mouse and Conventional
KO Mouse
Forward Genetic Approach
One of the origins of the clock genes discovery is
the studies using Drosophila. Although researchers
back then employed fruit flies as a simple model, it
took over a decade for scientists to identify the causal
gene period from period mutant flies and 13 more years
for them to find the human orthologue of drosophila
period gene (Konopka and Benzer, 1971; Reddy et al.,
1984; Zehring et al., 1984; Tei et al., 1997). Nowadays,
mammalian molecular clock machinery is welldescribed (Takahashi, 2017). Takahashi’s group
opened the door for mammalian clock genetics
through their success in forward genetic N-ethyl
N-nitrosourea (ENU) screening, identifying a rhythm
disorganized Clock mutant mouse and the causative

Clock gene (Vitaterna et al., 1994; King et al., 1997). It
is also noteworthy that mutagen-based screening
continues to generate new findings, as represented
by the discovery of Fbxl3 (Godinho et al., 2007;
Siepka et al., 2007). In the case of natural circadianrhythm mutant Tau hamster, it took over a decade
for the causative gene CKIε to be identified (Ralph
and Menaker, 1988; Lowrey et al., 2000). As it isuncommon to discover natural-born clock mutants,
mutagenesis screenings, due to their high efficiency,
have become a precursor of high-throughput in vivo
analysis.
Reverse Genetic Approach
Conventional KO Mouse. Through applying a genedriven approach, several genes were recognized asclock genes, which are the primary components ofthe molecular clock mechanism. For instance, thespecific functions of Per1-3 and Bmal1 genes in vivo
were confirmed by generating conventional embryonic stem cell (ESC)-based KO mice (Zheng et al.,
1999; Bunger et al., 2000; Shearman et al., 2000; Baeet al., 2001; Cermakian et al., 2001; Zheng et al.,2001). Similarly, Cry1 and Cry2 were identified asclock genes through analyzing ES-based KO mice(van der Horst et al., 1999; Vitaterna et al., 1999). It
should be noted that a global collaboration to systematically knock out all genes in the mouse genome
promoted the reverse genetics approach (Guanet al., 2010). International projects, such as the Mutant
Mouse Resource & Research Center at UC Davis(https://mmrrc.ucdavis.edu/mmrrc-program/),
aim to prepare KO-ESCs and KO mice libraries
based on gene trapping, gene targeting, and/or
gene-editing technologies. The International Mouse
Phenotyping Consortium (IMPC) project (https://www.mousephenotype.org/) is creating a genomewide annotation of gene functions, which is done
through systematically developing and phenotyping a collection of standardized single-gene KO mice
(Beckers et al., 2009; de Angelis et al., 2015; Dickinson et al., 2016). In 2020, Zhang et al. analyzed theindirect calorimetry (IC) data set from over 2000
wild-type mice collected by IMPC and established amachine learning program. They screened mice from
750 mutant lines and identified 5 novel genes
involved in circadian misalignment (Zhang et al.,2020). Similarly, for-profit companies have KO micelibraries, and researchers can even order gene-modified animals online.
Conditional KO Mouse. Conditional KO mice are generated by various methods, including the Cre/LoxP
system. They have been utilized to study the spatiotemporal function of clock genes. Bmal1 is a suitableMinami et al. / High-throughput in vivo analysis in new era

target to study the spatio-temporal function of themolecular clock. Through a single-gene disruption,
the normal circadian rhythmicity can be abolished(Bunger et al., 2000). As a pioneering study, Storch
et al. (2007) developed mice with a conditional allele,
Bmal1, and analyzed the effect of retina-specific lossof Bmal1 function by crossing mice with a CHX10-Cre
transgene. Other groups also developed Bmal1 floxedmutant mice independently (Westgate et al., 2008;
Shimba et al., 2011; Ratajczak et al., 2012). Here, floxed
indicates a part of Bmal1 that is sandwiched by the
LoxP sites. By crossing the floxed mice with the celltype-specific Cre recombinase expressing mice,researchers performed various organ-specific loss ofclock function studies on organs including muscle,bone, cartilage, adrenal cortex, kidney, and immune
cells (Shimba et al., 2011; Gibbs et al., 2012; Takaradaet al., 2012; Dyar et al., 2014; Dumbell et al., 2016;
Nikolaeva et al., 2016; Takarada et al., 2017; Dan et al.,2020; Qian et al., 2020). In the brain, especially in the
suprachiasmatic nucleus (SCN) of the clock center,the development of conditional Bmal1 KO mouse
contributes to unveiling cell-type-specific molecular
clock functions (Mieda, 2019; Hastings et al., 2020).
Following are examples: GABA-neuron-specificBmal1 KO mouse, AVP-neuron-specific Bmal1 KO
mouse, and VIP-neuron-specific Bmal1 KO mouse(Mieda et al., 2015; Weaver et al., 2018; Todd et al.,2020). Along with the increasing demands on conditional KO tools, international repositories haveoffered such mice to the scientific community(McMurray et al., 2012; Murray et al., 2012; H. Lee,2021). One representative depository is the JAX Cre
Repository (https://www.jax.org). It offers morethan 300 Cre tool strains, including Cre expressing
strains, inducible Cre strains, Cre reporter strains,and loxP-flanked (floxed) strains (https://www.jax.
org/research-and-faculty/resources/cre-repository).

High-Throughput Screening in Vitro
Mammalian Cell-based Screening
Compared with animal studies, cell-based analysis
possesses its own notable advantages in terms ofscale, space, and speed. Molecular clock studies incultured mammalian cells began from the Rat-1 study
by Schibler’s group (Balsalobre et al., 1998). Since themolecular oscillators in the cultured cells that the
researchers used were identical to those in the SCN,
they could investigate molecular clock function utilizing cultured cell lines (Yagita et al., 2001).
Nowadays, numerous laboratories employ NIH3T3
fibroblast as a mouse clock model and U-2OS boneosteosarcoma as a human clock model (Akashi and3

Nishida, 2000; Vollmers et al., 2008; Isojima et al.,2009). Although the mammalian cell-based screening
is not the primary focus of this review, we introduced
it briefly here from a perspective of high-throughput
assays.
Genetic Perturbations
Supported by the development of bioinformatics
and laboratory automation, cell-based screening has
become available. Particularly, it consists of genomewide gene modification tools, including full-length
cDNA, shRNA, and siRNA libraries. Using U-2OS
cells, one group developed a siRNA-based circadianrhythm phenotype screening system based on a 384well plate (Vollmers et al., 2008). Maier et al. (2009)
tested 2660 RNAi constructs targeting kinases andphosphatases, and Zhang et al. (2009) knocked down
approximately 1000 genes genome-wide by RNAi so
as to investigate molecular clock components/modifiers. From the time when clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR
associated protein (Cas) 9 appeared, modifying the
genomes of cell lines became easier. In terms of clock
genes, lipofection-based Csnk1d and Csnk1e KO
NIH3T3 cells, lentivirus-based Fbxl3 KO U-2OS cell,and adenovirus-based Bmal1 and Per2 KO U-2OS cellwere reported (Korge et al., 2015; Guo et al., 2019; Jinet al., 2019). Notably, gene-editing based KO strategy
will enable researchers to perform genome-wide KO
analysis in vitro in the near future. Details of the
CRISPR/Cas method will be discussed later.
Chemical Perturbations
Compared with genetic perturbations, chemical
perturbations are more suitable for cell-based highthroughput analysis. In chronobiology, the concept ofchemical biology appeared as early as in 2007, alongwith the many successful cases reported afterward(Liu et al., 2007). Two groups tested 1280 chemicals ina library of pharmacologically active compounds(LOPAC) for their effects on circadian period length(Hirota et al., 2008; Isojima et al., 2009). Through thestudy, our group succeeded to identify that temperature-insensitive phosphorylation of CKIδ defines
period length. Shinohara et al. (2017) further identified aurintricarboxylic acid (ATA) as a dephosphorylation-specific inhibitor of CKIδ using LOPAC1280
and studied how temperature-independent phosphorylation reactions were achieved. Kay, Hirota,
and their colleagues promoted a chemical-biologicalapproach. They successfully identified small circadian rhythm modifying molecules utilizing U-2OS
systems. These molecules include Longdaysisn,

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which targets CKIδ, CKIα, and ERK, LH846, which
inhibits CKIδ specifically, and KL001, which prevents
the ubiquitin-dependent degradation of CRYs (Hirota
et al., 2010; Lee et al., 2011; Hirota et al., 2012).
Moreover, Chen et al. (2012) performed a highthroughput screening of ~200,000 synthetic compounds using the fibroblasts from adult mouse’s ear,which harbored Per2::LucSV fusion gene, and found11 circadian period and amplitude modifiers. He
et al. (2016) screened an in-house collection of 5300
components using the heterozygous ClockΔ19/+
PER2::Luc reporter cells, which exhibited sustained
reporter rhythms with low amplitudes, and discovered the circadian rhythm amplitude enhancer,
Nobiletin. Reviews on various small circadian rhythm
modifiers are available elsewhere (Hirota and Kay,
2015; Gloston et al., 2017; Chen et al., 2018; Miller and
Hirota, 2020).

Carroll, 2016). CRISPR/Cas9 system is composed of
Cas9 DNA endonuclease, CRISPR RNA (crRNA), andtrans-activating crRNA (tracrRNA). This tracrRNAtypically binds to the crRNA and forms a ribonucleoprotein complex with Cas9. The crRNA then guides
the Cas9 complex to the target sequence (Deltcheva
et al., 2011). This 2-component RNA system was further simplified to synthetic single-guide RNA(sgRNA) consisting of a fusion of crRNA andtracrRNA. Today, the CRISPR/Cas9 system is widely
applied in genome editing for knocking out genes(Adli, 2018). For example, introducing the Cas9mRNA with 1 sgRNA into a zygote can efficiently
generate mosaic mice containing KO cells. As statedin the Scientific Background part of the Nobel Prize in
Chemistry 2020, “genomic manipulations are no longer an experimental bottleneck.”
Crossing Remains a Bottleneck

The Bottlenecks for High-Throughput
Screening in Vivo
Two Bottlenecks of Genetically Modified Mice
Although forward genetic screenings were the pioneer of high-throughput analysis in vivo, it took years
for such screenings to develop from the separation ofmutants to the identification of causal genes. Reverse
genetics altered the situation, but the generation andthe preparation of gene-modified animals for testing
phenotype remained laborious and time-consuming.
The difficulties for increasing the experimental scales
and experimental speeds exist in the genome manipulation and the crossing steps (Susaki et al., 2017)(Figure 1a).
“Genomic Manipulations Are No Longer anExperimental Bottleneck”
Conventionally, a mutation needs to be introduced
to the ESCs in order to produce a KO or KI mouse,
which is a laborious process. A specific gene is either
disrupted or inserted through gene targeting basedon the spontaneous homologous recombination,
which occurs at low frequencies (Austin et al., 2004;
Capecchi, 2005). However, the genomic manipulation
step has become easier due to the appearance ofgenome-editing technologies. Such technologies rely
on DNA double-strand break repair mechanisms andhave been applied in the forms of TALEN(Transcription activator-like effector nuclease), ZFN(Zinc finger nucleases), and CRISPR/Cas-based
methods (Gaj et al., 2013; Chandrasegaran andIn terms of the conventional KO/KI mice, the
KO/KI-ESCs are injected into the wild-type blastocysts. This process gives rise to the chimera micedeveloped from both the ESCs and the wild-type
host’s embryo-derived cells. The obtained chimera
mice (F0) are crossed with the wild-type mice, andtheir F1 generation carries a mutated genome if
germline transmission occurs. According to
Mendel’s Laws, a homozygous mutant is expected
to appear in the F2 generation. This suggests that atleast 9 months (3 months/generation x 3 generations) are needed to obtain the first homozygous
mutant. Furthermore, the resulting mutants often
need to backcross to some inbred strains, such as the
C57BL/6, several times if the mutants are generatedusing the F1 hybrids or the 129 strain derived ESCs.
These crossing steps cannot be abbreviated andremain a problem (Figure 1b). In contrast to thelaborious methods for producing conventional KI/
KO mice, the CRISPR/Cas9-based genome edited
mice require no such backcrossing if they are generated based on a desired inbred genetic background.
Because using multiple sgRNAs simultaneously can
introduce mutations in various genes, KO mice withmultiple dysfunctional genes can be obtained without crossing (Wang et al., 2013). However, in thiscase, the founder mice (F0) often contain a mosaic ofwild-type and KO cells, and the rate of generatingcomplete biallelic mutant mice is relatively low.
Thus, though they could avoid the backcrossing
issue, the mice generated using the CRISPR/Cas9
technology still consume time in experiments
because in real practice, expanding colonies bycrossing would still be necessary to obtain enoughmice to analyze, and this takes time.

Minami et al. / High-throughput in vivo analysis in new era(a)Before CRISPR(b) Genetic Modification

(c)

5

D

(targeting)

HTA

ESCs

CRISPR with sgRNAs

ESCs

CRISPR
8-stageblastocyst

embryos

pronuclei/
cytoplasm
Chimera

HTA

mouse
Crossing

Crossing

Crossingm/+
Triple CRISPR

HTA

Crossing

m/m

m/m

biallelic KO
Conventional KO/KI mouse Triple CRISPR

ES Mouse

Figure 1. A conceptual illustration of next-generation mammalian genetics. (a) Three bottles analogy. Before the development of
CRISPR technology, the bottle (high-throughput analysis in vivo; HTA) was sealed tightly (top), but the CRISPR technology had
increased the efficiency of genetic modification in mice and thus loosened the stopper in the bottle. The Triple CRISPR method eliminated the need to cross, which is illustrated as pulling the stopper fully from the bottle (bottom). (b-d) Steps of gene modification animal
production in conventional KO/KI mice (b), Triple CRISPR (c), and ES mice (d). Note that the crossing step is necessary for KO/KI mice.
F0 chimera mice are crossed to wild-type mice and the F1 heterozygotes (m/+) are intercrossed to obtain the homozygotes (m/m). If ESCs
are derived from the hybrids or the 129 strains, a further backcrossing step is needed (~10 generations) (b). In cases of the Triple CRISPR
and ES mice, the F0 mice can be used for analysis and no further crossing needed (c-d). Details are described in the main texts. Squareindicates the original gene and triangle indicates the mutated gene. Abbreviations: CRISPR = clustered regulatory interspaced shortpalindromic repeat; KO = knock out; KI = knock in; ESCs = embryonic stem cells; HTA = High-throughput analysis.

Next-Generation Mammalian Genetics
Genetics Without Crossing
To increase the throughput of analysis in vivo andpropel mammalian genetics to the next generation,
we must tackle the “crossing” issue. We define nextgeneration mammalian genetics as the “genetics
without crossing” (Susaki et al., 2017; Ukai et al.,2017, 2019). This can be achieved through efficient
KO/KI methods that generate analyzable KO/KI
mice within one generation. There are 2 possibilities:
generating whole-body biallelic mutants with almost
100% probability using the CRISPR/Cas9 method (orother gene-editing methods), or generating micealmost entirely from ESCs derived cells. From an ethical viewpoint, next-generation genetics contributes
to reducing the number of experimental animals per

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study. Because the timing of the birth of F0 mutants
depends on the experimental schedule, it is possible
to systematically obtain animals that are matched inages and genders. In addition, researchers do not
obtain heterozygous mutants that are useful for mating but are normally useless in experiments (Figure
1c and 1d).
Triple-CRISPR Method
Triple-targeted CRISPR (Triple CRISPR) is amethod that employs 3 kinds of sgRNA simultaneously targeting 1 gene. From a computational analysis, Sunagawa et al. found that a multiple-target
CRISPR strategy is more efficient than a single-targeted CRISPR, which is consistent with the experimental results from a dual-target CRISPR case (Zhouet al., 2014). Experimentally, the biallelic KO efficiencies of a single gene (Tyr) using each of the 3 sgRNAs
was 36.0%, 54.2%, and 64.7%, respectively. However,through Triple CRISPR, the biallelic KO efficiency of
the Tyr gene increased to 97.5%, which was muchhigher than the minimum computational estimation,
82.6%. This increase was probably due to the irreversible mutations resulting from the Triple CRISPR. It isof note that the increased efficiency was reproducible
by using other sets of 3 sgRNAs. Furthermore, thepossibility of the off-target effects could be practicallyeliminated by comparing phenotypes of KO mice
resulting from 2 different sets of 3 sgRNAs (Sunagawa
et al., 2016).
Triple CRISPR has enabled us to perform genedriven phenotype analysis at the F0 generation. Thisadvancement allows us to implement high-throughput in vivo analyses, such as a comprehensive KO
mice study of 1 gene family at a single laboratorylevel (Figure 2a). Following are some examples fromprevious sleep research, which employed the tripleCRISPR technology. Sunagawa et al. (2016) generated
and analyzed the sleep phenotypes of KO mice with
mutations in the Tyr, Bmal1, Hcrt, and NMDA receptors (5 genes). Tatsuki et al. (2016) found the importance of intracellular calcium concentration in sleep
regulation by generating KOs of Ca2+-dependent K+
channels (8 genes), voltage gated Ca2+ channel α1
subunits (10 genes), plasma membrane Ca2+ ATPases(4 genes), and calcium/calmodulin-dependent protein kinase type II (4 genes). Niwa et al. (2018) identified muscarinic acetylcholine receptors, Chrm1 andChrm3, as the essential genes for rapid eye movement(REM) sleep by generating and analyzing a total of 16
KO mice with dysfunctional nicotinic acetylcholine
receptor (11 genes) and muscarinic acetylcholine
receptors (5 genes). Yoshida et al. (2018) studied theimportance of the leak potassium channel. They identified Kcnk9 as an essential regulator of sleep lengthby producing and examining KOs of leak potassiumchannel genes (14 genes).
The ES Mouse
The ES KO Mouse. The ES mouse is almost entirely
ESC-derived and carries mutations throughout the
body in its F0 generation (Figure 1d). The tetraploid complementation method was first applied
to generate the ES mouse (Duncan, 2005). The otherapproach was the microinjection of ESCs to 8-cellstage embryos (Poueymirou et al., 2007; DeChiara
et al., 2009, 2010). Huang et al. (2008) produced ES
mice via ESCs injection to 4- or 8-cell embryos.
ESCs are typically maintained with leukemia inhibitory factor (LIF) and serum (or BMP) to keep theirundifferentiated states. LIF can activate STAT3,
which additionally triggers Sox3 and Nanog. All
these further activate Oct3/4, keeping the pluripotency of the ESCs (Niwa et al., 2009). Moreover, theERK inhibitor, FGF receptor inhibitor, and GSK3
inhibitor (3i) enable efficient derivation and germline-competent propagation (Ying et al., 2008).
Kiyonari et al. (2010) established and maintained
C57BL/6 N derived ESC line with serum-free
medium supplemented 3i, and they succeeded in
producing the ES mouse. As an example, the feasibility of Kiyonari’s system in gene KO mice wasshown in Chrm1/Chrm3 double KO mice production (Niwa et al., 2018). Although the ES KO miceare powerful tools, their importance pales in comparison to what can be done with the CRISPR editing. Only a limited number of groups still use thistechnique now.
The ES KI Mouse. The value of ES mice could beshown in the generation of KI mice. The reliability
and robustness of the ES KI mouse method were
witnessed in several studies (Susaki et al., 2014;
Tainaka et al., 2014; Wang et al., 2016). Ukai et al.
have improved the efficiency of the KI ESCs production, producing various types of KI ESCs in parallel(Ode et al., 2017; Ukai et al., 2017). Furthermore,combining their procedure with the ES mouse technology (Kiyonari et al., 2010), systematic productionof diverse kinds of KI ES mice in parallel wasachieved using a short period of time (2-3 months)(Ukai et al., 2019).
Loss-of-function and gain-of-function studies arenecessary pairs to show causal relationships between
gene(s) and phenotype. Antoch et al. illustrated acausal relationship between the behavioral phenotype of Clock mutant mouse and ClockΔ19 mutation via
an in vivo complementation study. In this research,
they generated TG mice harboring bacterial artificial

Minami et al. / High-throughput in vivo analysis in new era

7(a)Gene family
Triple CRISPR
KO mice series
Phenotypeanalysis(b)

Modification sites

Gene
KO-rescue mice
(ES mouse)

Phenotypeanalysis(c)

AAV triple CRISPR, other virus based CRISPR...

Bmal1 floxed

Series of cell type specific Cre drivers

other floxed
Systematicconditional

Peripheral tissues
liver

kidneymuscle

eye

SCN
Other

AVP cells VIP cells NMS cells GRP cells Other cells

KO mouse
Figure 2. Comprehensive system-level analyses achieved by or will be achieved by next-generation mammalian genetics. (a) A comprehensive KO mice analysis covering the whole 1 gene family. In this review, we showed examples of KO mice covering almost all genefamily, which includes Nr2s, Nr3s, Kcns, Cacna1s, Atp2bs, Camk2s, Chrnas, Chrnbs, Chrms, and Kcnks (Sunagawa et al., 2016; Tatsuki
et al., 2016; Niwa et al., 2018; Yoshida et al., 2018). (b) A comprehensive gene structure–level analysis by KO-KI rescue using ES mouse.
By changing the modification sites systematically (such as the phosphorylation sites) and introducing the gene-of-interest KO-ESCs,
the corresponding series of KO-rescue ESCs/mice can be generated. In this review, the Cry1 phosphorylation sites were discussed as
an example (Ode et al., 2017). (c) A systematic generation of conditional KO mice by the high-efficacy Cre/loxP system and/or the AAVTriple-CRISPR technology. The expansion of Cre driver lines and/or the development of a virus-based CRISPR/Cas9 method will enable
us to generate systematic conditional KO mice throughout all body/cell types. Abbreviations: KO = knock out; KI = knock in; ESCs
= embryonic stem cells; AAV = adeno-associated virus; CRISPR = clustered regulatory interspaced short palindromic repeat; SCN =suprachiasmatic nucleus; ES cells = embryonic stem cells; AVP = arginine vasopressin; VIP = vasoactive intestinal peptide; NMS =neuromedin-S; GRP = gastrin-releasing peptide.

chromosomes (BAC), including the wild-type Clockgene (Antoch et al., 1997). However, the inverse procedures called “KI and KO-rescue” remained arduousand expensive until recently. As the ES mouse technology enabled people to analyze various kinds of

KI-KO rescue mice in parallel during a short duration,
researchers were able to perform a high-throughput
KI and KO-rescue study for analyzing phenotypes ofvarious Cry1 mutants (Ode et al., 2017) (Figure 2b).
First, Cry1-/-, Cry2-/- ES mice were generated and8

JOURNAL OF BIOLOGICAL RHYTHMS / Month 202X

confirmed that they displayed no rhythmic behaviors
as previously reported in conventional KO mice (vander Horst et al., 1999; Vitaterna et al., 1999). Thisarrhythmic phenotype was then rescued by insertinga wild-type Cry1 gene in the ROSA26 sites. Moreover,
the authors selected 17 CRY1 mutations and added
these mutated CRY1 genes into different ESCs, which
lacked Cry genes. The mutant CRY1 rescue-ES mice
were further analyzed. Compare with the wild-types,
the Cry1(S243D)-rescued ES mouse displayed ashorter circadian rhythm behavior, whereas theCry1(S243A)-rescued ES mouse showed a longer circadian rhythm behavior (Ode et al., 2017). Recent
technological advances have enabled us to establish
KI mice using the CRISPR/Cas technique (see below).
The CRISPR/Cas-based technology, unlike ES mice, ismore commonly employed by research groups whengenerating KI-KO rescue mice.

Generation of Region-Specific Gene
Modification Animals in Short
DurationOne-step Conditional KO Mouse
Although a significant amount of information has
been obtained using the conventional KO/KI strategies, they generally lack spatial and temporal specificity, which can be improved by conditional KO
approaches. In the Cre/LoxP system case, researchers are required to obtain both a Cre driver mouseand a floxed gene-of-interest mouse first and thenmate them to obtain the desired conditional KO
mouse. Extensive efforts have been made to accelerate the process of sandwiching part of a gene at the
LoxP sites through the CRISPR/Cas-based KI strategy (Yang et al., 2013; Nakagawa et al., 2016;
Nishizono et al., 2020). CRISPR/Cas9-based highefficient KI technologies with electroporation havebeen reported and improved by many researchers(Horii et al., 2017; Miyasaka et al., 2018). A long singlestranded DNA (lss DNA) homology directed repair(HDR) approach sped up the generation of floxed
animals (Yoshimi et al., 2016; Codner et al., 2018).
Efficient addition with ssDNA inserts-CRISPR (EasiCRISPR) is one of such technologies, which uses an
lss DNA and Cas9 protein. Quadros et al. (2017) succeeded in generating 7 floxed mice and 6 KI micewith 8.5% to 100% and 25% to 67% success rates,
respectively. Ohtsuka et al. showed that genome
editing could be performed directly on fertilized
eggs in oviducts and called this technique theimproved-genome editing via oviductal nucleic
acids delivery (iGONAD) (Ohtsuka et al., 2018;
Gurumurthy et al., 2019). This method can also be

employed for generating conditional alleles (Shanget al., 2021). Using these technologies with oocytes
carrying tissue-specific Cre, 1-step generation of theconditional KO in F0 mouse could be accomplished(Horii et al., 2017; Miyasaka et al., 2018).
Cross-Species Studies Accelerated by
Next-Generation Mammalian Genetics
Humanized Animal Models
Humanized animals are defined as experimental
animal models xenotransplanted with human cells,
tissues, organs, and/or expressing human gene products (Walsh et al., 2017; Brendel et al., 2020; Moriwaki
et al., 2020; Stripecke et al., 2020). As a xenotransplantation model, humanized immune system mice, created from engrafting human stem cells to severely
immunodeficient mice, are well-known examples(McCune et al., 1988; Mosier et al., 1988; Shultz et al.,
1995; Ito et al., 2002; Allen et al., 2019). A genomicallyhumanized mouse is a mouse model produced byintroducing a human genomic region to a mouse, andit enables us to more comprehensively understandhuman health and diseases (Zhu et al., 2019; Moriwaki
et al., 2020). At first, researchers employed transgenic
mice with a piece of human genomic region. They
then utilized a KI mouse and replaced the genome
sequence with a human orthologous DNA (Devoy
et al., 2011). In 2001, Fu’s group identified a S662G
mutation in the human PER2 gene as a causal mutation for a kindred diagnosed with familial advanced
sleep phase syndrome (FASPS) (Toh et al., 2001).
Moreover, the group introduced a BAC harboring
either the wild-type or the mutated PER2s, including
the S662G mutation, to mice lacking the innate Per2
gene (Per2-/-). They discovered that the S662GPER2/Per2-/- BAC-TG mouse exhibited a shorter
behavioral rhythm in constant darkness (Y. Xu et al.,2007). Moreover, numerous coding and non-coding
human PER3 polymorphisms are associated withdiverse phenotypes and diseases (Archer et al., 2018).
Particularly, Hasan et al. generated 2 lines of humanized PER3 mice, which contained a human PER3
variable-number tandem-repeat (VNTR) insertion at
Per3 exon 17. It was concluded here that the PER3VNTR polymorphism modified the homeostatic
sleep response without significantly influencing circadian parameters (Hasan et al., 2014).
Improving the efficiency of humanized mouse production by KI and testing various types of KI mice inparallel are imperative steps in assessing the phenotypic diversity of diseases and/or evaluating the effects
of multiple candidates of disease-related single nucleotide polymorphisms (SNPs). Notably, gene-editing

Minami et al. / High-throughput in vivo analysis in new era

technology will accelerate the development of
KI-based-humanized mice. A KI method using singlestranded oligodeoxynucleotides (ssODNs) as a donor
DNA in genome-editing tools, such as the CRISPR/
Cas9, has become the most popular option in introducing an SNP (Bollen et al., 2018). In terms of middlerange insertion (a few hundred bp), KI methods thatuse lss DNA and/or double-strand DNA are employedby researchers (Miyasaka et al., 2018; Yao et al., 2018).
For long-range insertion (approximately 200 kb), suchas replacing an entire mouse gene with a human gene,a KI method utilizing 2 ssODNs is considered useful(Yoshimi et al., 2016). Currently these technologies arecommercially provided, and researchers can even orderhumanized animals online. Ukai et al. (2019) further
explained this humanized animals’ possibility in theirprevious review.
GWAS Studies for Considering Possible
Candidates of Humanization
Many mental illnesses, including Alzheimer’s disease, bipolar disorder, dysthymia, major depression,
Parkinson’s disease, psychosis, seasonal affective disorder, and schizophrenia, were found to be associatedwith clock genes, including BMAL1, CLOCK, CRY1,
CRY2, NR1D1, PER1, PER2, PER3, RORA, and RORB
(Jagannath et al., 2013, 2017). Thus, genetically modified mice harboring humanized clock genes with SNPs
could contribute to unveiling the real associations
between circadian clock machinery and psychiatricdiseases. CLOCK-3111 T/C (rs1801260) is one of thewell-described SNPs in the human CLOCK gene
(Schuch et al., 2018). It was first identified as an SNP
associated with diurnal preference and was further
found to be associated with sleep disorders (Katzenberg
et al., 1998; Serretti et al., 2005; Benedetti et al., 2007).
However, some reports revealed no association
between T3111C SNP and sleep phenotypes (Robilliard
et al., 2002; Chang et al., 2011). To our knowledge, thismutation has not been studied in vivo using humanized mice, but it has been tested in vitro using a Clock/mouse embryonic fibroblast (Ozburn et al., 2016).
Chronotypes, or diurnal preference, are suggested
as a polygenic basis of phenotypes. The first genomewide association study (GWAS) on chronotypes wasreported in 2007. The data obtained from 749 subjects
were analyzed, and several genetic loci with suggestive linkages to habitual bedtime were identified,including CSNK2A2, PROK2, and CLOCK (Gottlieb
et al., 2007). Kalmbach et al. reviewed the genetic
basis of human chronotypes based on 3 large-scale
GWASs; one of them comprised 89,283 samples of
23andMe and the other two included 100,420 and
128,266 samples respectively from the UK Biobank.
They pointed out that RGS16, PER2, FBXL3, and AK5

9were commonly recognized as the significant loci forchronotype through these 3 GWASs (Hu et al., 2016;
Jones et al., 2016; Lane et al., 2016; Kalmbach et al.,2017). In 2019, using the accelerometer data from85,670 UK Biobank participants, Jones et al. (2019)
performed a GWAS of 8 derived sleep traits representing sleep quality, quantity, and timing and identified 47 genetic associations, and RGS16 was also
included. Marukonen et al. (2020) investigated 8433
subjects from a Finnish general population. They discovered a novel genetic association between NR1D2(RevErbβ) and chronotypes, but their GWAS analysis
failed to show significant genome-wide associations.
Although the studies above all illustrated a clear relationship between human genes and chronotypes,
future researchers should keep in mind that significant loci varied in studies. As SNPs are also often correlated with nearby SNPs, discussing causality would
be hard. Nevertheless, testing such candidate humangenes and/or SNPs in mice in vivo would still provide valuable hints for understanding complicatedphenotypes like the human chronotypes.

Future Perspectives
Virus-based CRISPR
Different types of viral vectors have their own
advantages and disadvantages (Navabpour et al.,2020; Chuang et al., 2021). The adeno-associated virus(AAV)-based Triple CRISPR, which does not rely on
the employment of ES cells or fertilized eggs, is under
extensive investigation. Kim et al. developed a multiplexed CRISPR-Cas9 system in a single AAV to simultaneously knock out 3 clock genes, namely the CSAC
(CRISPR/Cas9-based single AAV system targeting thecircadian clock) (Kim et al., 2021). They constructed
multiplexed U6 promoter-driven sgRNA expression
cassettes for AAV packaging in which a maximum of 3
sgRNAs can be harbored. The researchers also developed CSAC-Pers targeting Per1 and Per2 simultaneously, CSAC-Crys targeting Cry1 and Cry2 at the sametime, and CSAC-Bmal1 targeting Bmal1. Furthermore,they unveiled that an infection of the CSACs to the
SCN reduced or abolished biological rhythms at bothtissue and organismal levels (Kim et al., 2021). Since
researchers can employ adult mice in these experiments, this virus-based approach is able to further
shorten the duration of mutant animal experiments.
Spatio-temporal Analysis
Although next-generation genetics we mentioned
above are powerful tools for generating systemic KOs

10

JOURNAL OF BIOLOGICAL RHYTHMS / Month 202X

and KIs, they still lack spatial and temporal specificities. Virus vectors enable us to introduce transgenes
to various cell types efficiently, including neurons.
Tso et al. injected AAV, 8-sgBmal, into the Aldh1 L1Cre mice’s bilateral SCN. They found that the Aldh1
L1-Bmal1 KO mice, lacking the SCN astrocytes clock,showed longer period of behavioral rhythms andPER2::Luc activity rhythms from cultured SCN slices(Tso et al., 2017). In combination with appropriate
surgical techniques (intracerebroventricular injections, the direct injection to the SCN, etc.), conditional
gene modification techniques (e.g., Cre-loxP system),and high-throughput gene modification tools (e.g.,
Triple CRISPR), the virus-based transfection system
will allow us to analyze cell-type-specific geneticallymodified animals in a very short duration (Figure 2c).
To evaluate cell-type specificity or time dependency
of used conditional gene modification, whole organ–level expression analysis is desirable. Previously, it
was laborious and unrealistic for researchers to makesections through the body and check the expression
patterns of target genes at a whole organ level.
Nowadays, however, scientists can use tissue-clearing technology to visualize and capture target gene
expression seamlessly with light-sheet microscopy.
Nonetheless, tissue-clearing technology itself is a
powerful tool for circuit-level and organ-level understanding (Pan et al., 2016; Gradinaru et al., 2018; Ueda
et al., 2020a, 2020b; Molbay et al., 2021).
Organism-level Systems Biology From Mice to
HumansSystems biology is a subfield of biology that understands life as integrated systems (Kitano, 2002). Life systems are composed of multi-layers such as genes,
mRNA, protein, cells, tissue, and organs. From the
beginning of the 21st century, systems biologicalapproaches have succeeded at molecular and cellularlevels. Organism-level systems biology might beachieved by next-generation genetics. At the same time,cross-sectional approaches between layers have beenhighlighted. Through combining gene and protein
structure-based analyses with cellular-level functional
assays, Xu et al. (2015) demonstrated that CRY1 competes for binding with coactivators to the intrinsically
unstructured C-terminal transactivation domain of
BMAL1 to establish a functional switch between activation and repression of CLOCK–BMAL1. Through a
combination of structure, cellular, and mouse studies,
our group succeeded in depicting a general picture of
the mammalian circadian period control by Cry1 (Odeet al., 2017). Recently, the influence of phosphorylation
on molecular timing was highlighted (Forger et al.,2007; Larrondo et al., 2015; Ode et al., 2017; Shinohara
et al., 2017; Narasimamurthy et al., 2018; Ode and Ueda,

2018; Masuda et al., 2020; Philpott et al., 2020;
Narasimamurthy and Virshup, 2021). Under such circumstances, these types of KI-KO mouse studies on
various clock genes could assist in portraying a pictureof a multidimensional real molecular clock.
Due to experimental and ethical limitations,researchers cannot perform the same experimental
approaches on humans as they have implemented on
mice. Humans have relatively long-life spans, diverse
genetic backgrounds, and different environmental
exposure histories, preventing them from becoming
ideal experimental subjects. Consequently, systems
biology in humans will be based on comparativeknowledge from other species, especially mice.
Plausible approaches could include performing alarge-scale screening in the human population to elucidate correlations between diseases and genomic
information, as discussed in the GWAS section.
Furthermore, experimental mice studies are imperative for testing causalities. These tests will be completed quantitatively, in parallel, and done in a timely
fashion. High-throughput analyses in vivo could
undoubtedly contribute to such occasions.

Conclusion
In this article, we have introduced the history ofanimal-based high-throughput analyses in chronological order. Although ENU mutagenesis screenings
showed their merits in their relatively high throughput, the identification step of these methods required along time to detect the causal genes. Regarding thisgene identification process, reverse genetics took lesstime, but it faced challenges in genome modificationand crossing. Recently, next-generation genetics,
which enabled researchers to evade the time-consuming crossing issue, emerged and shed light on highthroughput analyses. This method relied on the newly
developed technologies, such as the Triple CRISPR.
Some possible targets of this new method include
humanized animals carrying human genes. Overall,studies from the past decades have offered precious
experience and tools for developing ideal animal models. These efforts will ultimately contribute to unveiling the truth underlying human circadian rhythms.

Acknowledgments
We thank all the lab members at the University of Tokyoand RIKEN BDR. We apologize to researchers whose work
is not cited due to space limitations. This work was supported by grants from the Brain/MINDS JP21dm0207049,
Science and Technology Directorate Platform Program forMinami et al. / High-throughput in vivo analysis in new era
Advanced Biological Medicine JP21am0401011, AMEDCREST JP21gm0610006 (AMED/MEXT) (H.R.U.), Grantin-aid for Scientific Research (S) JP25221004 (JSPS
KAKENHI) (H.R.U.) and Scientific Research (C) JP20K07282(JSPS KAKENHI) (Y.M.), HFSP Research Grant Program
RGP0019/2018 (HFSP) (H.R.U.), ERATO JPMJER2001 (JST)(H.R.U.), MEXT Quantum Leap Flagship Program (MEXT
QLEAP) Grant Number JPMXS0120330644, and an intramural Grant-in-aid from the RIKEN BDR (H.R.U.). H.R.U.
is a co-inventor on a patent covering Triple CRISPR (PCT/
JP2015/086259 [pending], patent applicant is RIKEN, otherco-inventors are K. Sumiyama, G.A. Sunagawa, M. UkaiTadenuma, D. Perrin) and patent applications covering the
CUBIC reagents (PCT/JP2014/070618 [pending], patentapplicant is RIKEN, other co-inventors are E. A. Susaki and
K. Tainaka; PCT/JP2017/016410 [pending], patent applicant is RIKEN, other co-inventors are K. Tainaka and T.
Murakami) and a co-founder of CUBICStars Inc.

Conflict of Interest Statement
The author(s) have no potential conflicts of interest withrespect to the research, authorship, and/or publication ofthis article.

ORCID iD
Hiroki R. Ueda

https://orcid.org/0000-0001-8342-9176

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