Available online at www.sciencedirect.com

ScienceDirect
Next-generation human genetics for organism-levelsystems biology
Hideki Ukai1,2, Kenta Sumiyama3 and Hiroki R Ueda2,4,5Systems-biological approaches, such as comprehensive
identification and analysis of system components andnetworks, are necessary to understand design principles ofhuman physiology and pathology. Although reverse genetics
using mouse models have been used previously, it is a low
throughput method because of the need for repetitive crossing
to produce mice having all cells of the body with knock-out or
knock-in mutations. Moreover, there are often issues from theinterspecific gap between humans and mice. To overcome
these problems, high-throughput methods for producing
knock-out or knock-in mice are necessary. In this review, we
describe ‘next-generation’ human genetics, which can be
defined as high-throughput mammalian genetics withoutcrossing to knock out human-mouse ortholog genes or to
knock in genetically humanized mutations.
Addresses
1
ES-mouse/Virus Core, International Research Center for
Neurointelligence (WPI-IRCN), UTIAS, The University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
2
Laboratory for Synthetic Biology, RIKEN Center for Biosystems
Dynamics Research, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
3
Laboratory for Mouse Genetic Engineering, RIKEN Center for
Biosystems Dynamics Research, 1-3 Yamadaoka, Suita, Osaka 5650871, Japan
4
Department of Systems Pharmacology, Graduate School of Medicine,
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan
5
International Research Center for Neurointelligence (WPI-IRCN),UTIAS, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 1130033, Japan
Corresponding author: Ueda, Hiroki R (uedah-tky@umin.ac.jp)

Current Opinion in Biotechnology 2019, 58:137–145
This review comes from a themed issue on Systems biology
Edited by Maria Klapa and Yannis Androulakis

https://doi.org/10.1016/j.copbio.2019.03.003
0958-1669/ã 2018 Elsevier Inc. All rights reserved.

Introduction
Systems-biological approaches are necessary to understandcomplex and dynamic biological phenomena, which occur by
the interaction of multiple molecules and cells in mammalianorganisms [1,2]. Systems biology consists of a multi-stage
process beginning with (1) comprehensive identificationwww.sciencedirect.com

and (2) quantitative analysis of individual system components
and networks, which leads to the ability to (3) control existing
systems toward a desired state, and (4) design new systems
based on an understanding of the underlying structural anddynamic principles. Systems-biological approaches have beenused in studies to understand phenomena at the molecular-tocellular level in mammals [3]. However, the application of thesame approach toward understanding of organism-level phenomena in mammals has just begun [4,5], and approaches tounderstanding human physiology and pathology remain moredifficult challenge.
Linkage analysis and genome-wide analysis, such as familybased linkage analysis, Trio linkage analysis, and GenomeWide Association Study (GWAS), have been useful tools tofind genetic variants that correlate with monogenic (orMendelian) and complex diseases [6,7]. Moreover, in combination with next-generation sequencing techniques, it has
been possible to find variants, such as single nucleotide
polymorphisms (SNPs), structural variants (SVs), small
insertions or deletions (indels), frequencies (common torare), and regions (coding and non-coding) [6,8]. There
are well-established correlations between genes and humandiseases, but their causal relationships are still unclear.
Reverse genetics by gene knockout (KO) or knock-in (KI)is a powerful method to clarify the causal relationshipbetween genetic variation and phenotype in organisms[9]. Reverse genetics in mice was established using
embryonic stem cells (ESCs) [10–12]. However, theproduction of genetically modified mice is generally
low-throughput and requires tremendous time, space,and effort to obtain mice of sufficient quality and quantity
to use for experimental assays. One reason for the lowthroughput generation of mutant mice is the low efficiency of homologous recombination. Another reason isthe complicated crossing process, which can take several
months to years, and repeated back-crosses are needed toobtain complete KO or KI homozygotes in the inbred
genetic background. Using these conventional methods,
it is difficult to comprehensively identify and analyze
molecular properties, networks, and cellular circuits ofcomplex and dynamic biological processes in an organism.
For this reason, ‘next-generation’ mammalian genetics,
which can be defined as mammalian genetics withoutcrossing, using genome editing and developmental engineering is becoming popular [5,13].
The mouse is a useful model animal for reverse genetics.
However, in some cases, KO mice reproduce only a part of
Current Opinion in Biotechnology 2019, 58:137–145

138 Systems biology

the human disease phenotype, show a more severe effectthan human cases, or have no phenotype at all [14]. Evenin these situations, reverse genetics in humans is experimentally and ethically impossible, which makes miceexperiments using established genetic techniques a logical alternative. Therefore, the development of geneticallyhumanized mice that closely mimic the human condition[15] is needed. Technologies for the synthesis of genomesized DNA [16] and artificial chromosome vectors [17]have been developed in recent years, and the productionof genetically humanized mice is becoming easier. In thisreview, we discuss basic concepts and technologies fornext-generation human genetics, which can be used togenerate KO or KI mice without crossing.

Conventional gene-targeted mouseproduction
To produce KO or KI mice by conventional methods(Figure 1a), a mutation is first introduced into ESCs using
spontaneous homologous recombination, which usually
has a relatively low frequency. Next, the KO-ESCs or KIESCs are injected into wild-type blastocysts to produce
chimeric mice (F0) containing some cells derived fromthe introduced KO-ESCs or KI-ESCs. If the introduced
KO-ESCs or KI-ESCs contribute to the germline of thechimeric mice, heterozygous mutant mice can be produced in the next generation progeny (F1). Therefore, in
principle, homozygous mutant mice can be found in theF2 generation obtained about nine months after ESC
injection. However, in practice, because of the low efficiency of homologous recombination, a small contribution
rate of ESCs to the germline, and a low efficiency ofmating with inbred strains or among F1 and F2 mice,
there are difficulties and delays in obtaining the desired
genetic background for reliable analysis of the phenotype.
Therefore, the conventional method requires anextended period of time (several months to years), large
space, and huge labor. International efforts, such as theInternational Knockout Mouse Consortium and International Mouse Expression Consortium, perform part of thislabor for genome-wide production of mutant mice [18].
However, for organism-level systems biology, the establishment of next-generation mouse genetics withoutcrossing, which can reduce the labor to a scale that canbe executed by a single laboratory or even an individual
researcher, is necessary.

Production of KO mice of a human-mouseortholog gene without crossing
Production of KO mice of a human-mouse ortholog geneis the most straightforward approach in reverse genetics toinvestigate the function of a human gene. Genome editing using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9(Cas9) system is a widely used technology for gene KO[19]. Introduction of Cas9 mRNA and one sgRNA into azygote can quickly and efficiently generate mosaic mice
Current Opinion in Biotechnology 2019, 58:137–145

containing KO cells. Microinjection or electroporation ofribonucleoprotein (RNP: a complex of synthesized
crRNA, tracrRNA, and recombinant Cas9 protein) into
the zygote, especially at the early pronucleus stage considerably increases the proportion of non-mosaic variants[20,21]. Thus, a more efficient KO method is needed toobtain whole-body bi-allelic KO mice without crossing.
A recently developed triple-target CRISPR (TripleCRISPR) method [22] using three kinds of sgRNA simultaneously targeting one gene, can generate whole-bodybi-allelic KO mice with almost 100% probability (Figures
1b, 2b, and 2d). In the case of tyrosinase gene KO,
efficiency of obtaining complete bi-allelic KO was>95% by using three sgRNAs simultaneously, comparedwith the low efficiency of 50% using single sgRNA.
Moreover, the high efficiency was reproducible by using
other sets of three sgRNAs [22]. Exome sequencing
results for both tyrosinase and Nr3a KO animals revealed
that there is no apparent sign of off-target cleavages [22],possibly because sgRNAs are designed to minimize theoff-target effect and Cas9 cleavage is only transiently
active around one to four cell stage due to the usage ofCas9 mRNA. Furthermore, by utilizing two sets of independent sgRNA trios for KO animal phenoscreening, thepossibility of off-target effects was eliminated [22]. The
efficient identification of critical genes for NREM sleep[22–24] and essential genes for REM sleep [25] by the
Triple-CRISPR system demonstrated the efficacy of thismethod. An open database of the set of triple-sgRNAtargets (http://crispr.riken.jp/) covering approximately
80% of all the genes in the mouse is a useful tool forcomprehensive identification and analysis of components
of a system.
Unlike the Triple-CRISPR method to introduce the
CRISPR/Cas9 system into a zygote, an in vivo (in oviduct)
electroporation (i-GONAD) method [26] does not
require isolation of zygotes from donor mice and transplantation into recipient mice. Therefore, a combinationof the Triple-CRISPR and i-GONAD methods may beable to further reduce labor and the number of miceneeded for the production of the desired KO mice.

Production of KI mice of a human-mouseortholog gene without crossing
To reproduce human SNPs and SVs in a mouse, various
genome-editing methods have been used on the zygote.
For the introduction of one SNP, the use of base editors[27–29] may be useful because direct editing of the basewithout a DNA double-strand break avoids generation of
unintended indels. Base editing in mouse embryos [30]and human embryos [31] has been successful. However,for reproducing several SNPs within a narrow range ofseveral tens of bases, a KI method using single stranded
oligodeoxynucleotides (ssODNs) as donor DNA in
genome-editing tools, such as CRISPR/Cas9, TALEN,
www.sciencedirect.com

Next-generation human genetics for organism-level systems biology Ukai, Sumiyama and Ueda 139

Figure 1(a)∼1 month∼ 3 months∼ 1 year (∼ 3 months/generation)

Gene targetingin ESCs

Injection of ESCs andtransplantation into a uterus

Crossing

129-derived ESC(LIF + serum)

129 strain

× 5-10 generations

ESCs injection
into a blastocyst

F0 chimera

Heterozygous
mutantHeterozygous
mutant(congenic)

Homozygous
mutant(congenic)

×

×

×

Animal for analysis

Inbred strain
for backcrossInbred strain
for backcrossHeterozygous
mutant(congenic)

Targeting(dependent on
spontaneous HDR)

~ 3 months(b)

Genome editing in zygotes andtransplantation into an oviduct

CRISPR
with sgRNAs

Inbred strain(c)Single gene
DSBs-induced
NHEJ
Injection into Bi-allelic KO mouse
Zygotepronuclei
or cytoplasm Animal for analysis~ 1 month~ 3 months

Genome editingin ESCInjection of ESCs andtransplantation into a uterus

Inbred strainderived ESC(3i + LIF)

Inbred strain

DSB

ESC injection
into 8-cell stageembryos

KI-ES mouse
Animal for analysis

TargetingSite-specific (dependent on
nuclease DSB-induced HDR)
Current Opinion in Biotechnology

Conventional and next-generation methods for mutant mouse production.
(a) A typical procedure for conventional targeted KO or KI mouse production. An inbred strain such as C57BL/6 is widely used for analysis.
However, hybrid or other inbred strains are used in the production stages for practical reasons. Therefore, in many cases, backcrossing isrepeated to generate congenic mice. In addition, inefficient gene targeting depending on spontaneous homologous recombination in ESCs
increases time and labor. (b) Next-generation KO mouse production and (c) KI mouse production. The use of inbred strain-derived zygotes or
ESCs, efficient genome editing in zygotes, and one-step production of whole body KI-ES mouse by 8-cell-stage embryo injection eliminate alldifficulties associated with crossing procedures. These F0 animals can be used in subsequent phenotyping experiments. In addition, efficient genetargeting using site-specific endonucleases in ESCs eliminates unnecessary labor.

and zinc-finger nuclease (ZFN), has been the most used
option [32]. To introduce multiple SNPs into a broad
range of the genome or reproduce SV, insertion of a larger
DNA fragment is necessary. Therefore, methods usingwww.sciencedirect.com

long ssODN [32], double-stranded DNA [33], or both
simultaneously [34] as donor DNA have been used.
However, the efficiency of the precise introduction oflarge fragments by homologous recombination is still not
Current Opinion in Biotechnology 2019, 58:137–145

140 Systems biology

Figure 2(a)Single-target CRISPR(b)

Triple-target CRISPRsgRNA: 50 ng/μL,
Cas9 mRNA: 100 ng/μL
CRISPR
with sgRNAs

3 sgRNAs: 50 ng/μ L each,
Cas9 mRNA: 100 ng/μL
CRISPR
with sgRNAs

DSB

Tyrosinase
gene

DSBs

Injection intocytoplasm
C57BL/6 strain(c)

ZygoteTyrosinase
gene
Injection intocytoplasm

C57BL/6 strain

Zygote(d)Current Opinion in Biotechnology

One-step generation of KO mice.
We designed a tyr gene KO experiment by single-target CRISPR (a) and Triple-CRISPR (b) in the C57BL/6 strain. Tyr is a gene coding fortyrosinase, which is an enzyme responsible for black coat color. A typical result of the experiment by single-target CRISPR (c) and Triple-CRISPR(d). The Triple-CRISPR method efficiently produces 100% bi-allelic KO littermates with white coat color. All animal experiments were approved by
the Institutional Animal Care and Use Committee of RIKEN Kobe Branch, and all animal care was in accordance with the Institutional Guidelines.

high enough to produce KI mouse without crossing. In
contrast to the low KI efficiency in the zygote (up to6.5%), the introduction of DNA into the embryo at thetwo-cell stage has high KI efficiency (up to 35%) [35].
However, first generation mice obtained by these methods are often mosaic. Thus, applying these methods to thezygote or embryo is not the best solution to produce KI
mice without crossing at present.
Genome-editing methods have also been successfully
applied to ESCs to generate KI ESCs with high efficiency(almost 90%) [13]. Advanced culture methods of ESCs
using three kinds of inhibitors (3i: SU5402 for FGFR,PD184352 for ERK, and CHIR99 021 for GSK3) andleukemia inhibitory factor (LIF) was efficient for establishing and culturing germline-competent ESCs, even if
the mouse supplying embryos was the inbred mouse suchas BALB/C and C57BL/6 (hereafter denoted as B6) [36].
The stable establishment and maintenance of ESCs
derived from B6 in ES mouse production are particularly
critical for the next-generation mammalian genetics.
Although B6 is the most standard strain in mouse genetics, B6-ESCs have limitations of less efficient chimera
Current Opinion in Biotechnology 2019, 58:137–145

formation and germ-line transmission, difficult maintenance, and genomic instability in standard culture conditions. However, the injection of 3i-medium-cultivated
KI ESCs into an 8-cell stage embryo was able to directly
and efficiently produce fully ESC-derived mice (ESmice) with all the cells of the body carrying KI mutations(contamination of the host embryo cell was 0.1% or less)even after many passages for the KI process [13,36,37].
Live-birth rates of the F0 ES mice reached 11–29% (% ofES mice/embryos transferred). Therefore, 30 (the bestcase) to 100 host embryos were enough for generating asufficient number (10) of ES mice for phenotyping.
These technologies can produce many KI mice with lesslabor and time and without the complexities of crossing[13,37] (Figure 1c).
Compared with zygote and embryo-based methods, theES-based production of KI mice may allow for more
complex genetic engineering, such as introducing SNPs
into multiple genes. Furthermore, ESCs allow for ease ofpreservation, re-examination, and selection of the desired
sex. The current mainstream method to produce ES miceis an 8-cell-stage injection method requiring advanced
www.sciencedirect.com

Next-generation human genetics for organism-level systems biology Ukai, Sumiyama and Ueda 141

technology and expensive equipment. However, devices
that can generate ES mice easily by aggregation with 8cell stage embryos were also developed [38]. These
devices may further improve throughput in the future.
Thus, KI ES-based production of KI mice without crossing is an effective method for organism-level systems
biology to analyze human disease-related genes.

chromosomes has been difficult. To overcome this problem, a method using germline stem cells (GSC) was
developed [44]. MAC-containing GSCs microinjected
into the seminiferous tubules of infertile recipients successfully produced mice containing MAC in cells in thewhole body without crossing. This method is suitable forthe production of mice containing human-specific genes.

Next-generation mouse genetics with agenetically humanized mouse

In contrast, to substitute some mouse genes or systems on
the host chromosomes with human genes or systems on
the artificial chromosome, a KO-rescue approach [37]
combined with a bi-allelic KO of the corresponding
mouse gene is necessary (Figure 3). However, a GSCbased method alone cannot accomplish this. Furthermore, a single phenotype in humans can be the resultof the interaction of multiple genes [45], which suggeststhat the simultaneous KO of multiple mouse genes may
be necessary for humanization of the system. These
highlight the many difficulties involved in replacing
mouse genes or systems with that of a human at GSC
and zygote-levels.

Mice that are genetically engineered based on genetic
variations in humans can be useful for various researchstudies, such as the elucidation of human disease mechanisms and the development of therapeutic methods.
However, there are only few proteins in which the aminoacid sequence is 100% conserved between human andmouse [39]. The differences in the sequences of mostorthologs may lead to differences in the expected phenotypes. Indeed, the phenotype observed in KO mice isoften not similar to that in humans [14]. Furthermore,some reports have suggested that a gene unique tohumans is involved in human diseases, such as psychiatricdisorders [40,41]. In these cases, reproducing the phenotype in mice may not be possible because no counterpart
gene is present. Therefore, it may be crucial to makegenetically humanized mice [15] in which the mousegenes or systems are replaced by the human genes orsystems (Figure 3).
Because genetic variations in humans occur not only inthe coding sequence but also in the non-coding sequence,
the DNA fragments introduced into the mouse forhumanization can be extremely long. Recently, de novo
synthesis of chromosomal-level long DNA has become
feasible [16]. Advanced synthetic biological technology
may be useful for the preparation of long DNA that is
responsible for a set of gene locus involved in any humantype system or quantitative trait loci (QTL).
Classically, artificial vectors, such as YAC/BAC/PAC,
have been used for preparing and transferring extremely
long DNA fragments [42]. Random or site-specific insertion into the zygote of long-DNA fragments necessary forhumanization may produce mice in a short period compared with insertion into ESCs. However, generation ofmosaics may be a problem. Moreover, random integrationof genes may occasionally destroy endogenous genes,
which complicates phenotypic analysis.
Another approach for humanization is the use of artificial
chromosomes, such as HAC/MAC [17], which does not
require insertion into a host chromosome. Humanizeddrug-metabolizing model mice were produced using artificial chromosome technology [43]. However, artificial
chromosomes often fall out during the crossing processof chimeric mice produced by conventional methods.
Thus, stable production of mice containing artificial
www.sciencedirect.com

Multiple rounds of genome editing are possible in one ESC
clone [46], such as numerous KOs of related mouse orthologs or further modification of introduced human genes[47] after the introduction of an artificial chromosome
carrying human genes or systems (Figure 3). In addition,using a method for mouse production without crossing,such as the ES-mouse method, there may be no need toworry about loss of the HAC or MAC during crossing. Thus,
production of a genetically humanized mouse by combining chromosome-level DNA synthesis and manipulation
technology, highly efficient genome editing techniques,and ES-mouse production without crossing is a usefultechnique for next-generation human genetics.
In contrast, researchers should carefully consider towhat extent potential mosaicism (e.g. mutational variations in the triple-CRISPR method, or undetectablecontamination of wild-type cells in the ES mousemethod) would affect the final results of a scientific study.
In our previous experiments, the phenotypic variations ofF0 mice were comparable with suitable control animals,suggesting that mutational variation/unwanted contamination of wild-type cells do not seem problematic at leasein these cases [22,37]. Even if an unfortunate unwanted
mutational variation, including the very rare off-target
effect, is transmitted to the germline, such variation
would not significantly affect the phenotyping under
the concept of ‘without crossing.’ To further exclude
the possibility of artifact phenotypes due to mosaicism,
we recommend the researchers to independently generate the KO/KI mice by using the second set of tripleCRISPR for the same gene, or by using an independent
clone of ES cells. Realizing such stringent criteria is
feasible in the next-generation genetics because it only
takes a few months.
Current Opinion in Biotechnology 2019, 58:137–145

142 Systems biology

Figure 3

Highly efficient genome editing technology(~ 1 month)
Inbred strain-derived ESC (3i + LIF)

ESC

Wild-type mouse genes/systems

ESC injection
into 8-cell stageembryos

Wild-type mouse

ssODN, lssODN, or
Taegeting vector

ZygoteESC

SNP
Modified mouse genes/systems

Modified (KI) mouse

ESC

Bi-allelic KO mouse genes/systems

Bi-allelic KO mouse

ESC

Bi-allelic KO mouse genes/systems+
Human genes/systems

Genetically humanized(KO-rescue) mouse

Triple-target
CRISPR

Genetic modification of mouse-ortholog genes

Inbred strain

ES-mouse technology(~ 3 month)

Artificial chromosome

ssODN, lssODN, or
Targeting vector

ESC

SNP
Bi-allelic KO mouse genes/systems+
Modified human genes/systems

Modifiedgenetically humanized(KO-rescue) mouse

Genetic modification of human-ortholog genes

Targeting vector
or

Current Opinion in Biotechnology

Next-generation genetics to generate genome-edited humanized mouse.
ES-mouse technology can generate KI mice with modified (SNP/CNV introduced) mouse genes without mating. Introduction of human-derived
genes/systems into bi-allelic KO ESCs prepared using Triple-CRISPR technology by KI or artificial chromosome-mediated transfer can produce
genetically humanized ESCs. Moreover, further modifications (introduction of SNP/CNV) in humanized ESCs are possible. ES-mouse technology
can also make genetically humanized mice from humanized ESCs without mating. Different colors of mice indicate that each mouse is of adifferent genotype.

Current Opinion in Biotechnology 2019, 58:137–145

www.sciencedirect.com

Next-generation human genetics for organism-level systems biology Ukai, Sumiyama and Ueda 143

The next-generation genetics also contributes to‘Reduction’ of 3R. In the next-generation methods, only
the F0 littermates of non-KO or non-ES mice are sacrificed, and no further animal is needed. In the conventional method, dozens of littermates are produced andsacrificed during the crossing procedure to select micewith an expected genotype. The number exponentially
increases when a more complicated genetic background(e.g. reproducing several SNPs) is needed in the conventional method, while the number of used animals is notdependent on the genetic complexity in the next-generation genetics. Thus, the next-generation techniques
reduce the overall animal usage by the biomedical
research community.

Conclusions
Comprehensive identification and analysis of systemcomponents and networks are necessary to understandthe design principles of complex and dynamic biologicalphenomena in organisms. ‘Next-generation’ mammalian
genetics for the direct production of KO or KI mice fromzygotes or ESCs without crossing, such as TripleCRISPR and ES-mouse methods, will facilitate the identification and the analysis of molecular networks andcellular circuits in organisms. Compared to the TripleCRISPR method to produce whole-body KO mice fromzygotes, the ES-mouse method has lower throughput.
Therefore, additional development of technologies basedon the concept of the next-generation genetics is needed
to improve the throughput of the ES-mouse method of KI
mice production.
Organism-level systems biology is coming to fruition withthe development of next-generation methods for mouseproduction, genomics, and phenotype analysis. The discovery of disease-correlated genetic variants by GWAS
and whole exome sequencing following the developmentof next-generation sequencing enables comprehensive
identification of system components. In addition,
improvements in whole-body clearing and imaging methods with single-cell resolution provide comprehensive
and quantitative experimental data at cellular resolution
on an organism scale [4,48,49]. Furthermore, the development of a high-throughput and non-invasive method
for phenotyping [22] will also be an attractive direction.
Organism-level systems biology based on these technologies will accelerate our understanding of complex anddynamic molecular and cellular circuits in humans.

Conflict of interest statement
Nothing declared.

Funding
This work was supported by the World Premier International Research Center Initiative (WPI), MEXT, Japan(H.U and H.R.U), a Grant-in-Aid for Scientific Research(B; JSPS KAKENHI grant number 18H02490; K.S.), a
www.sciencedirect.com

Grant-in-Aid for Scientific Research on Innovative Areas(JSPS KAKENHI grant number 15H05949 to K.S.),
AMED-CREST (AMED/MEXT, H.R.U.), CREST(JST/MEXT, H.R.U.); Brain/MINDS (AMED/MEXT,
H.R.U.); the Basic Science and Platform Technology
Program for Innovative Biological Medicine (AMED/
MEXT, H.R.U.); Grant-in-Aid for Scientific Research(S) (JSPS KAKENHI grant 25221004 and 18H05270 to
H.R.U.); Grant-in-Aid from Takeda Science Foundation(H.R.U.).

Acknowledgements
We thank our laboratory members at the RIKEN Center for Biosystems
Dynamics Research (BDR) and the University of Tokyo, in particular, N.
Hori, E. Matsushita, Y. Uranyu, N. Matsumoto, N. Nakai for their kind help
in preparing the materials and supporting experiments. We also thank Dr.
Hiroshi Kiyonari and Dr. Arthur Millius, RIKEN BDR for helpful advice.

References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
 of outstanding interest
1.

Kitano H: Systems biology: a brief overview. Science 2002,
295:1662-1664.

2.

Kitano H: Computational systems biology. Nature 2002,
420:206-210.

3.

Ukai H, Ueda HR: Systems biology of mammalian circadianclocks. Annu Rev Physiol 2010, 72:579-603.

4.

Susaki EA, Ueda HR: Whole-body and whole-organ clearing
and imaging techniques with single-cell resolution: toward
organism-level systems biology in mammals. Cell Chem Biol
2016, 23:137-157.

5.


Susaki EA, Ukai H, Ueda HR: Next-generation mammalian
genetics toward organism-level systems biology. NPJ Syst Biol
Appl 2017, 3:15.
A comprehensive review on organism-level systems biology by mammalian genetics without crossing.
6.

Londin E, Yadav P, Surrey S, Kricka LJ, Fortina P: Use of linkage
analysis, genome-wide association studies, and nextgeneration sequencing in the identification of diseasecausing mutations. In Pharmacogenomics: Methods andProtocols. Edited by Innocenti F, van Schaik RHN. Totowa, NJ:
Humana Press; 2013:127-146.

7.

Manolio TA, Collins FS, Cox NJ, Goldstein DB, Hindorff LA,
Hunter DJ, McCarthy MI, Ramos EM, Cardon LR, Chakravarti A
et al.: Finding the missing heritability of complex diseases.
Nature 2009, 461:747-753.

8.

Kilpinen H, Barrett JC: How next-generation sequencing istransforming complex disease genetics. Trends Genet 2013,
29:23-30.

9.

Jaenisch R: Transgenic animals. Science 1988, 240:1468-1474.

10. Evans MJ, Kaufman MH: Establishment in culture ofpluripotential cells from mouse embryos. Nature 1981,
292:154-156.
11. Martin GR: Isolation of a pluripotent cell line from early mouseembryos cultured in medium conditioned by teratocarcinoma
stem cells. Proc Natl Acad Sci U S A 1981, 78:7634-7638.
12. Capecchi MR: Gene targeting in mice: functional analysis of the
mammalian genome for the twenty-first century. Nat Rev Genet
2005, 6:507-512.
13. Ukai H, Kiyonari H, Ueda HR: Production of knock-in mice in asingle generation from embryonic stem cells. Nat Protoc 2017,12:2513-2530.
Current Opinion in Biotechnology 2019, 58:137–145

144 Systems biology

14. Elsea SH, Lucas RE: The mousetrap: what we can learn whenthe mouse model does not mimic the human disease. ILAR J
2002, 43:66-79.
15. Devoy A, Bunton-Stasyshyn RK, Tybulewicz VL, Smith AJ,
Fisher EM: Genomically humanized mice: technologies andpromises. Nat Rev Genet 2011, 13:14-20.
16. Boeke JD, Church G, Hessel A, Kelley NJ, Arkin A, Cai Y,
Carlson R, Chakravarti A, Cornish VW, Holt L et al.: Genome
engineering. The genome project-write. Science 2016,
353:126-127.

32. Bollen Y, Post J, Koo BK, Snippert HJG: How to create state-ofthe-art genetic model systems: strategies for optimal crisprmediated genome editing. Nucleic Acids Res 2018,
46:6435-6454.
33. Yao X, Zhang M, Wang X, Ying W, Hu X, Dai P, Meng F, Shi L,
Sun Y, Yao N et al.: Tild-crispr allows for efficient and precise
gene knockin in mouse and human cells. Dev Cell 2018, 45:526536 e525.
34. Yoshimi K, Kunihiro Y, Kaneko T, Nagahora H, Voigt B, Mashimo T:
Ssodn-mediated knock-in with crispr-cas for large genomic
regions in zygotes. Nat Commun 2016, 7:10431.

17. Oshimura M, Uno N, Kazuki Y, Katoh M, Inoue T: A pathway fromchromosome transfer to engineering resulting in human andmouse artificial chromosomes for a variety of applications tobio-medical challenges. Chromosome Res 2015, 23:111-133.

35. Gu B, Posfai E, Rossant J: Efficient generation of targeted large
insertions by microinjection into two-cell-stage mouseembryos. Nat Biotechnol 2018, 36:632-637.

18. Meehan TF, Conte N, West DB, Jacobsen JO, Mason J, Warren J,
Chen CK, Tudose I, Relac M, Matthews P et al.: Disease model
discovery from 3,328 gene knockouts by the international
mouse phenotyping consortium. Nat Genet 2017,49:1231-1238.

36. Kiyonari H, Kaneko M, Abe S, Aizawa S: Three inhibitors of fgf
receptor, erk, and gsk3 establishes germline-competentembryonic stem cells of c57bl/6n mouse strain with highefficiency and stability. Genesis (New York, NY: 2000) 2010,
48:317-327.

19. Adli M: The crispr tool kit for genome editing and beyond. Nat
Commun 2018, 9:1911.

37. Ode KL, Ukai H, Susaki EA, Narumi R, Matsumoto K, Hara J,
Koide N, Abe T, Kanemaki MT, Kiyonari H, Ueda HR: Knockout
rescue embryonic stem cell-derived mouse reveals circadianperiod control by quality and quantity of cry1. Mol Cell 2017,65:176-190.
The article demonstrated the efficacy of genetics without crossing bygenerating 20 strains of ES mice in a short period to conduct comprehensive characterization of molecular properties in an organism.

20. Aida T, Chiyo K, Usami T, Ishikubo H, Imahashi R, Wada Y,
Tanaka KF, Sakuma T, Yamamoto T, Tanaka K: Cloning-free
crispr/cas system facilitates functional cassette knock-in inmice. Genome Biol 2015, 16:87.
21. Hashimoto M, Yamashita Y, Takemoto T: Electroporation of cas9
protein/sgrna into early pronuclear zygotes generates nonmosaic mutants in the mouse. Dev Biol 2016, 418:1-9.
22. Sunagawa GA, Sumiyama K, Ukai-Tadenuma M, Perrin D,
Fujishima H, Ukai H, Nishimura O, Shi S, Ohno RI, Narumi R et al.:Mammalian reverse genetics without crossing reveals nr3a asa short-sleeper gene. Cell Rep 2016, 14:662-677.

38. Sumiyama K, Matsumoto N, Garcon-Yoshida J, Ukai H, Ueda HR,Tanaka Y: Easy and efficient production of completely
embryonic-stem-cell-derived mice using a micro-aggregation
device. PLoS One 2018, 13:e0203056.
In this study, a microdevice for ESCs and 8-cell embryo aggregation wasdevised that could be used in future massively paralleled aggregation
system for next-generation genetics.

23. Tatsuki F, Sunagawa GA, Shi S, Susaki EA, Yukinaga H, Perrin D,
Sumiyama K, Ukai-Tadenuma M, Fujishima H, Ohno R et al.:Involvement of Ca2+-dependent hyperpolarization in sleep
duration in mammals. Neuron 2016, 90:70-85.

39. Waterston RH, Lindblad-Toh K, Birney E, Rogers J, Abril JF,
Agarwal P, Agarwala R, Ainscough R, Alexandersson M, An P
et al.: Initial sequencing and comparative analysis of themouse genome. Nature 2002, 420:520-562.

24. Yoshida K, Shi S, Ukai-Tadenuma M, Fujishima H, Ohno RI,
Ueda HR: Leak potassium channels regulate sleep duration.
Proc Natl Acad Sci U S A 2018, 115:E9459-E9468.

40. Fiddes IT, Lodewijk GA, Mooring M, Bosworth CM, Ewing AD,
Mantalas GL, Novak AM, van den Bout A, Bishara A,
Rosenkrantz JL et al.: Human-specific notch2nl genes affectnotch signaling and cortical neurogenesis. Cell 2018,
173:1356-1369.e1322.

25. Niwa Y, Kanda GN, Yamada RG, Shi S, Sunagawa GA, UkaiTadenuma M, Fujishima H, Matsumoto N, Masumoto KH,
Nagano M et al.: Muscarinic acetylcholine receptors chrm1 andchrm3 are essential for rem sleep. Cell Rep 2018, 24:2231-2247.
e2237.
26. Ohtsuka M, Sato M, Miura H, Takabayashi S, Matsuyama M,
 Koyano T, Arifin N, Nakamura S, Wada K, Gurumurthy CB: Igonad: a robust method for in situ germline genome
engineering using crispr nucleases. Genome Biol 2018, 19:25.
The authors generated genome-edited mice by directly electroporating
genome editing components into zygotes in situ.
27. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR: Programmable
editing of a target base in genomic DNA without doublestranded DNA cleavage. Nature 2016, 533:420-424.
28. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH,
Bryson DI, Liu DR: Programmable base editing of a*t to g*c ingenomic DNA without DNA cleavage. Nature 2017,
551:464-471.
29. Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M,
Mochizuki M, Miyabe A, Araki M, Hara KY et al.: Targeted
nucleotide editing using hybrid prokaryotic and vertebrate
adaptive immune systems. Science 2016, 353.
30. Sasaguri H, Nagata K, Sekiguchi M, Fujioka R, Matsuba Y,
Hashimoto S, Sato K, Kurup D, Yokota T, Saido TC: Introductionof pathogenic mutations into the mouse psen1 gene by baseeditor and target-aid. Nat Commun 2018, 9:2892.
31. Liang P, Ding C, Sun H, Xie X, Xu Y, Zhang X, Sun Y, Xiong Y,
Ma W, Liu Y et al.: Correction of beta-thalassemia mutant bybase editor in human embryos. Protein Cell 2017, 8:811-822.
Current Opinion in Biotechnology 2019, 58:137–145

41. Suzuki IK, Gacquer D, Van Heurck R, Kumar D, Wojno M, Bilheu A,
Herpoel A, Lambert N, Cheron J, Polleux F et al.: Human-specificnotch2nl genes expand cortical neurogenesis through delta/
notch regulation. Cell 2018, 173:1370-1384.e1316.
42. Giraldo P, Montoliu L: Size matters: use of yacs, bacs and pacs
in transgenic animals. Transgenic Res 2001, 10:83-103.
43. Kazuki Y, Kobayashi K, Aueviriyavit S, Oshima T, Kuroiwa Y,
Tsukazaki Y, Senda N, Kawakami H, Ohtsuki S, Abe S et al.:Trans-chromosomic mice containing a human cyp3a cluster
for prediction of xenobiotic metabolism in humans. Hum Mol
Genet 2013, 22:578-592.
44. Shinohara T, Kazuki K, Ogonuki N, Morimoto H, Matoba S,
 Hiramatsu K, Honma K, Suzuki T, Hara T, Ogura A et al.: Transfer
of a mouse artificial chromosome into spermatogonial stem
cells generates transchromosomic mice. Stem Cell Rep 2017,
9:1180-1191.
GS cell-mediated stable transfer of an artificial chromosome into offspring was achieved.
45. Gandal MJ, Leppa V, Won H, Parikshak NN, Geschwind DH: Theroad to precision psychiatry: translating genetics into diseasemechanisms. Nat Neurosci 2016, 19:1397-1407.
46. Stacey A, Schnieke A, McWhir J, Cooper J, Colman A, Melton DW:
Use of double-replacement gene targeting to replace themurine alpha-lactalbumin gene with its human counterpart inembryonic stem cells and mice. Mol Cell Biol 1994,
14:1009-1016.
47. Abe S, Kobayashi K, Oji A, Sakuma T, Kazuki K, Takehara S,
Nakamura K, Okada A, Tsukazaki Y, Senda N et al.: Modificationwww.sciencedirect.com

Next-generation human genetics for organism-level systems biology Ukai, Sumiyama and Ueda 145

of single-nucleotide polymorphism in a fully humanized cyp3amouse by genome editing technology. Sci Reports 2017,
7:15189.
SNP modification of CYP3A5 on the CYP3A-MAC by genome editing
technology using the CRISPR/Cas9 system was successfully performed
in both mouse ES cells carrying the CYP3A-MAC and fertilized eggs
carrying the CYP3A-MAC.

dimensional single-cell-resolution whole-brain atlas using
cubic-x expansion microscopy and tissue clearing. Nat
Neurosci 2018, 21:625-637.
49. Tainaka K, Murakami TC, Susaki EA, Shimizu C, Saito R,
Takahashi K, Hayashi-Takagi A, Sekiya H, Arima Y, Nojima S et al.:Chemical landscape for tissue clearing based on hydrophilic
reagents. Cell Rep 2018, 24:2196-2210.e2199.

48. Murakami TC, Mano T, Saikawa S, Horiguchi SA, Shigeta D,
Baba K, Sekiya H, Shimizu Y, Tanaka KF, Kiyonari H et al.: A three-www.sciencedirect.com

Current Opinion in Biotechnology 2019, 58:137–145