Human Genetics
https://doi.org/10.1007/s00439-020-02198-4

ORIGINAL INVESTIGATION

Combi‑CRISPR: combination of NHEJ and HDR provides efficientand precise plasmid‑based knock‑ins in mice and rats
Kazuto Yoshimi1,2 · Yuichiro Oka3,4 · Yoshiki Miyasaka5 · Yuko Kotani5 · Misato Yasumura3 · Yoshihiro Uno5 ·Kosuke Hattori5 · Arisa Tanigawa5 · Makoto Sato3,4 · Manami Oya6 · Kazuhiro Nakamura6 · Natsuki Matsushita7 ·Kazuto Kobayashi8 · Tomoji Mashimo1,2,5,9Received: 29 February 2020 / Accepted: 19 June 2020
© The Author(s) 2020

Abstract
CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway,
which is dominant in zygotes, efficiently induces insertion or deletion (indel) mutations as gene knockouts at targeted sites,
whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a doublestranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome
sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted
in 20–33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site thatwas designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp,
such as EGFP, mCherry, Cre, and genes of interest, at the other exon site. These findings indicate that this combinatorial
method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA
cassettes in mice and rats.

Background
Zinc finger nucleases (ZFNs), transcription activator-like
effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) enable rapid and precise genetic
manipulation in mammalian cells (Gaj et al. 2013; Peng
Electronic supplementary material The online version of thisarticle (https​://doi.org/10.1007/s0043​9-020-02198​-4) contains
supplementary material, which is available to authorized users.

5

Institute of Experimental Animal Sciences, Graduate Schoolof Medicine, Osaka University, Osaka 565‑0871, Japan

6

Department of Integrative Physiology, Nagoya University
Graduate School of Medicine, Nagoya 466‑8550, Japan

7

Division of Genome Engineering, Center for Experimental
Medicine and Systems Biology, Institute of Medical Science,
The University of Tokyo, Tokyo 108‑8639, Japan

Division of Laboratory Animal Research, Aichi Medical
University School of Medicine, Aichi 480‑1195, Japan

8

Department of Anatomy and Neurosciences, Graduate Schoolof Medicine, Osaka University, Osaka 565‑0871, Japan

Department of Molecular Genetics, Institute of Biomedical
Sciences, Fukushima Medical University Schoolof Medicine, Fukushima 960‑1295, Japan

9

Genome Editing Research and Development Center,
Graduate School of Medicine, Osaka University,
Osaka 565‑0871, Japan* Tomoji Mashimo
mashimo@ims.u‑tokyo.ac.jp
1

2

3

4

et al. 2014). Recently, the introduction of the CRISPR-Cas9
system has enabled the knockout of genes in zygotes via
non-homologous end-joining (NHEJ) with unprecedented
simplicity and speed (Mashimo 2014; Peng et al. 2014;
Sander and Joung 2014; Wang et al. 2013). Multiple geneknockouts can also be achieved using several different sgRNAs designed to target multiple genes (Wang et al. 2013;
Yoshimi et al. 2014). More recently, several groups, including ours, reported genome engineering using the zygote
electroporation of CRISPR-Cas9, which represents an easy

Division of Animal Genetics, Laboratory Animal ResearchCenter, Institute of Medical Science, The Universityof Tokyo, Tokyo 108‑8639, Japan

Department of Child Development, United Graduate Schoolof Child Development, Osaka University, Osaka 565‑0871,
Japan

13

Vol.:(0123456789)

Human Genetics

a

ATG

d

TCA

exon 1

12

13

14

2A

lssDNA

iCre

90 bp

477 bpHR
2A ERT2

lssDNAiCre

ERT2

297 bp57 bp

sgRNA-2
sgRNA-1
GTTGTCTAGCAGGTATTAAGTGG CCTGTTATTTATCAAGGTTCCAC

HR

Mc4r

sgRNA-1
sgRNA-2
AAAGGACTATAGATCATAAGGGTATAAATGACTGCTTAATGTGG

Kcnab1genome

-210

-55

1

exon

genomeHR
3.0 kbp

1

996

1,305

NHEJ
1.1 kbp

0.8 kbp

dsDNA(vector)

0.7 kbp

dsDNA(vector)

TAA

HR

87

NHEJ
0.3 kbp

ATG

0.7 kbp

TAA

TGA

Mc4r-F
iCre

Kcnab1-F

KI

CDS 2A ERT2 iCre ERT2KI

3’ UTR

iCre

CDS

2A

iCre

3’ UTR
Mc4r-R

Kcnab1-R

WTSpacer#1 TATCGCAAGTATAAATGACTGCTTATTATGTGGACTCCATATCGCAAGTATAAATGACTGCTTAAATGTGGACTCCA#2#5GGATCTATTAT------(∆57bp)-------ACTCCAb

STOP PAM

STOP PAM

PAM

TATCGCAAGTATAAATGACTGCTTAATGTGGACTCCAWT GATCATAAGGCAATGCA#1 CAGCTTGAGGCAATGCAWT TTCTTTCCTGTTATTTATCAAGGTTCCACACTTGT

GGATGGGGACTAAGTGGGGG#3 TAGTGT-------(∆83bp)-------ACACTTGT#2#3#2 CAGCTTGAGGCAATGCA#5 CAGCTTGAGGCAATGCA#41-cell2-cellpups
pups
KI pups
sgRNA embryo transfer delivered KO-indels
(%)
(n)
(n)
(n)
(%)

e

PAM

WT TAGCAGGTATTAAGTGGGGG
GGATGGGGACTAAGTGGGGG#2 TTCTTTCCTGTT---TATCAAGGTTCCACACTTGT

GGATGGGGACTAAGTGGGGG#4 TTCTTTCCTGTT---TATCAAGGTTCCACACTTGT

1-cell2-cellpups
pups
KI pups
sgRNA embryo transfer delivered KO-indels
(%)
(n)
(n)
(n)
(%)

HR

#1

48

29

6

4 (67)

0

HR

#1

193

112

17

13 (76)0

lssDNA

#1

182

154

23

17 (74)0

lssDNA

#1

139

111

21

20 (95)

0

138

66

9

6 (67)

3 (33)

185

101

9

9 (100)3 (33)

Combi-CRISPR #1&#2c㻝 㻞 㻟 㻠 㻡 㻢 㻣 㻤 㻹㻌㻥

Combi-CRISPR #1&#2f㻹 㻝 㻞 㻟 㻠 㻡 㻢 㻣 㻤 㻥 㼃 㻼

1 screening

1 screening

iCre

iCre

2nd screening

2nd screeningst

st

Upstream

Downstream㻝 㻞 㻟 㻠 㻡 㻢 㻣 㻤 㻹㻌㻥㻝 㻞 㻟 㻠 㻡 㻢 㻣 㻤 㻥

13Upstream

Downstream㻹 㻝 㻞 㻟 㻠 㻡 㻢 㻣 㻤 㻥 㻹 㻝 㻞 㻟 㻠 㻡 㻢 㻣 㻤 㻥Human Genetics◂Fig. 1  Injection of two sgRNAs, Cas9, and a donor dsDNA into

mouse zygotes. a Methods to integrate the P2A-ERT2-iCre-ERT2
cassette at the terminal codon of the Kcnab1 gene with lssDNA(above) or dsDNA (bottom). Microinjection of two sgRNAs, Cas9,
and dsDNA provided three KI mice (#1, 2, and 5) carrying precise
KIs of the iCre cassette at the sgRNA-1 targeting site and insertion or deletion mutations at the sgRNA-2 targeting site. b, e Comparison of three methods using dsDNA with single sgRNA-1 (HR),lssDNA with sgRNA-1 (lssDNA), or dsDNA with two sgRNAs(Combi-CRISPR) for KIs in mouse zygotes. c, f PCR analysis using
primer sets amplifying the internal region of the iCre cassette (firstscreening) or for 5′ genome-donor boundary (Upstream) and donor3′ genome boundary (Downstream in second screening) in delivered mouse pups (#1–9 for c and #1–5 for f). M: 100 bp DNA ladder marker. d Methods to integrate P2A-iCre cassette at the terminalcodon of the Mc4r gene with lssDNA (above) or dsDNA (bottom).
Microinjection of two sgRNA, Cas9, and dsDNA provided three KI
mice (#2–4) carrying precise KIs of the iCre cassette at the sgRNA-1
targeting site and several deletion mutations at the sgRNA-2 targetingsite

and rapid alternative to the elaborate pronuclear injection
procedure for genome editing in mice and rats (Kaneko et al.
2014; Qin et al. 2015).
CRISPR-Cas9-mediated knock-ins (KIs) in zygotes havebeen achieved via homology-directed repair (HDR) with adonor DNA template (Yang et al. 2013). Either microinjection or electroporation of CRISPR-Cas9 together with asingle-stranded oligodeoxynucleotide (ssODN) has becomea widely used method to introduce point mutations or short
tag sequences in zygotes (Inui et al. 2014; Yang et al. 2013).
The use of long ssDNA (lssDNA) has been developed as anefficient alternative donor template for the CRISPR-Cas9mediated KIs of cassette sequences or two loxP sites (Miuraet al. 2015; Yoshimi et al. 2016). Quadros et al. reported
Easi-CRISPR for creating KI or conditional knockout mousemodels using lssDNA produced by in vitro transcription
and reverse transcription or obtained from the company(Quadros et al. 2017). We also reported a CLICK method
using lssDNA purified from nicked dsDNA plasmids forthe manipulation of GFP cassette sequences (Yoshimi et al.
2016), or for the quick generation of conditional knockout
mice (Miyasaka et al. 2018). However, these approaches provide less efficiency or incomplete KIs when more than 2 kb
sequences of lssDNA are used as a donor template.
Using double-stranded DNA (dsDNA) as a donor template, the HDR-mediated KI efficiency is usually very low inzygotes. A cloning-free method, the direct nuclear deliveryof Cas9 protein complex with chemically synthesized dualRNAs, was investigated for the efficient generation of knockin mice (Aida et al. 2015). Several approaches to improve
the HDR efficiency include chemical reagents (Song et al.
2016) or small molecules (Maruyama et al. 2015). Other
approaches have used the stabilization of ssODNs (Renaud
et al. 2016) and sgRNAs (Hendel et al. 2015) by chemical
modification. Several other technical approaches, including

homology-independent targeted integration (HITI) (Suzuki
et al. 2016), obligate ligation-gated recombination (ObLiGaRe) (Auer et al. 2014; Maresca et al. 2013), and precise
integration into target chromosome (PITCH) (Nakade et al.
2014) have been reported, some of which were efficient incultured or in vivo cells. Recently, homology-mediated endjoining (HMEJ) (Yao et al. 2017) and targeted integration
with linearized dsDNA (Tild)-CRISPR (Yao et al. 2018)
were shown to provide efficient gene KIs in mouse andhuman cells, but some of these methods were not efficientin zygotes, or have not been well evaluated. Recently, twocell homologous recombination (2C-HR), a method basedon introducing CRISPR reagents into embryos at the twocell stage, has been reported as an efficient gene-integration
approach in zygotes (Gu et al. 2018).
In this study, we report a new powerful method of generating plasmid-based KI in mice and rats using the CRISPRCas9 system. The principle concept of this method is the
combination of highly efficient editing via NHEJ and thelow efficiency, but precise editing of HDR in zygotes. We
termed this approach Combi-CRISPR, and the combinationof NHEJ and HDR provides efficient and precise knock-ins
of large DNA fragments in mice and rats. A similar concept
to our approach was recently reported whereby the combination of NHEJ and HDR (termed SATI) efficiently integrated
transgenes in a targeted manner (Suzuki et al. 2019). Our
approach has improved the SATI method further by delivering a second sgRNA to enhance HDR.

Results
Generation of plasmid‑based knock‑in micewith CRISPR‑Cas9 and two sgRNAs
sgRNA-1 was designed at the terminal codon of the potassium voltage-gated channel subfamily A member regulatory beta subunit 1 (Kcnab1) gene to integrate a bi-cistronic
expression cassette encoding tamoxifen-inducible Crerecombinase (Fig. 1a and Supplementary Table 1). We
prepared a dsDNA donor vector including a 2.9 kbp P2AERT2-iCre-ERT2 cassette with a 297 bp 5′ homology arm(HA) and a 695 bp 3′ HA, or lssDNA donor with a 297 bp 5′
HA and a 57 bp 3′ HA, which was purified according to ourpreviously reported method (Yoshimi et al. 2016) (Fig. 1a).
We injected dsDNA (3 ng/µl) or lssDNA (40 ng/µl) with
Cas9 mRNA (20 ng/µl) and sgRNA-1 (25 ng/µl) into mouse
C57BL/6 embryos, which resulted in 4 of 6 delivered pups
and 17 of 23 delivered pups, respectively, carrying indel
mutations at the sgRNA-1 target site (Fig. 1b). However,no KI mouse with dsDNA or lssDNA was obtained via conventional HDR.

13

Human Genetics

To increase the knock-in efficiency, we designed an additional sgRNA-2 within intron 13 (55 bp upstream of exon
14) to cut the second site of the genomic region and thehomologous region of the donor dsDNA plasmid (Fig. 1aand Supplementary Table 1). Injection of Cas9 mRNA
(50 ng/µl), two sgRNAs (sgRNA-1 and sgRNA-2, 25 ng/µl each), and the donor dsDNA (1 ng/µl) into C57BL/6
embryos resulted in nine delivered pups. First screening by
PCR and sequencing with primers amplifying the sgRNA-1
target region and the internal region of the iCre cassette(Supplementary Table 2) revealed six pups carrying indel
mutations (KO) and five pups carrying a P2A-ERT2-iCreERT2 insertion (Fig. 1c). Second PCR and sequencing analysis using primer sets for the 5′ genome-donor boundaryand donor-3′ genome boundary identified three KI mice thatcarried indel mutations, such as a 2-bp insertion or 57 bp
deletion at the intron region (Upstream) and precise KI of
the P2A-ERT2-iCre-ERT2 cassette at the terminal codon of
the Kcnab1 gene (Downstream; Fig. 1a–c and Supplementary Table 2).
Crossing the KI founder mouse with a wild-type C57BL/6
mouse confirmed the germline transmission of the KI allelein F1 KI mice (Supplementary Fig. 1a). To further evaluate whether the ERT2-iCre-ERT2 allele was functional,
we crossed the founder KI mice with reporter mice (B6.
Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J: Jackson 007914),
which provided inducible recombination events at the flox
site and tdTomato expression in the neurons of F1 KI mice
(Supplementary Fig. 1b, c).

Combination of the NHEJ and HDR pathways
to induce efficient knock‑ins
To duplicate this KI approach, we targeted the melanocortin 4 receptor (Mc4r) gene with the T2A-iCre bi-cistronic
expressing vector. We designed sgRNA-1 targeting the terminal codon of the Mc4r gene and sgRNA-2 cutting boththe 3′ untranslated region (UTR) and the 696 bp 3′ HA ofthe donor plasmid (Fig. 1d and Supplementary Table 1).
Table 1  Generation of knock-in
mice with CRISPR-Cas9

Generation of KI rats using the Combi‑CRISPR
methodTo examine whether the Combi-CRISPR method could beapplied to rat zygotes, we targeted the terminal exon of theTargeting

MethodVector size (bp)

Eggs

Two-cell

Pups

KO

KI

Ctgf-ERT2-iCre-ERT2

lssDNAHR
Combi-CRISPR
lssDNA
Combi-CRISPR
lssDNA
Combi-CRISPR
Combi-CRISPR
Combi-CRISPR
Combi-CRISPR

3345
4563
4563
1493
2662
1685
2984
5232
4136
3459

177
50
151
997
197
340
165
165
246
239

151
38
59
898
68
327
117
114
130
136

18
7
6
220
6
71
22
9
17
10

13
ND
ND
42
4
38
14
6
10
6

0
0
1
1
1
0
2
2
1
2

Slc12a1-iCre
Bmi1-mCherry
Plxnd1-ERT2-iCre-ERT2
Cdkn2a-tdT-DTR
Cdkn2a-CD2-DTR

13

Injection of sgRNA-1, sgRNA-2 (25 ng/µl each), Cas9mRNA (20 ng/µl), and the donor vector (2 ng/µl) into
C57BL/6 mouse embryos resulted in nine pups, five of
which carried the iCre cassette at the first screening (Fig. 1e,f). Second PCR and sequencing with the boundary primer
sets (Supplementary Table 2) revealed three mice carrying
indel mutations at the sgRNA-2 targeting site and a precise
KI allele of the P2A-iCre cassette at the sgRNA-2 targetingsite (Fig. 1d–f). We also repeated the other approaches using
a conventional dsDNA targeting vector or lssDNA donor
template with sgRNA-1/Cas9. However, no KI mouse wasobtained among similar numbers of delivered pups by these
two methods (Fig. 1e).
To evaluate the efficiency of this method using two sgRNAs/dsDNA compared with the other methods, we targeted five other genomic loci and six different donor vectors from 2.6 to 5.2 kbp in size including Cre, inducible
Cre, mCherry, and diphtheria toxin receptor (DTR) genes(Table 1). Overall, nine KI mice were obtained among 70
delivered pups, indicating 13% efficiency, although no KI
mouse was identified when using the lssDNA methods. In
the KI mice generated by the two sgRNAs method, we often
observed various indel mutations at the sgRNA-2 targetingsites that were designed in the intron region. We also foundprecise KI alleles at the sgRNA-1 targeting sites that were
designed at the terminal codon of each gene. Therefore, fromthe sequence data of the KI founder mice, we speculated thatindel mutations at the sgRNA-2 targeting sites were repaired
via NHEJ and KI alleles were precisely repaired via HDR
between the sgRNA-1 targeting genome and the donor plasmid (Fig. 3). We termed this KI method using two sgRNAs/
Cas9 and a dsDNA donor Combi-CRISPR (combination of
NHEJ and HR repair pathway to induce efficient knock-ins).

Human Genetics

parvalbumin (Pvalb) gene and tyrosine hydroxylase (Th)gene to integrate a 1.7 kbp P2A-Cre cassette for the bi-cistronic expression of Cre-recombinase in rats. We prepared
dsDNA donor vectors with 1–1.5 kbps HAs or lssDNA with
60–300 bps HAs for the two genes (Fig. 2a, d). We also prepared sgRNA-1 targeting the terminal codon of the Pvalb
or Th gene and sgRNA-2 targeting the intron upstream of
the terminal exon. First, we examined the lssDNA method
for the two genes. Microinjection or electroporation of lssDNA and Cas9/sgRNA-1 into F344 rat embryos resulted
in 47 pups with the Pvalb gene and 37 pups with the Th
gene (Fig. 2b, e). Overall, 26 KO pups for the Pvalb geneand 27 pups for the Th gene were obtained, but no KI rat
was identified by PCR analysis for the two genes. However,
the microinjection of dsDNA, Cas9, and two sgRNA-1 andsgRNA-2, into 197 F344 rat embryos for the Pvalb geneand 182 embryos for the Th gene resulted in two and six
pups delivered, respectively. Of these, one (#2) and three(#3, #4 and #5) KIs were obtained, respectively, by the first
PCR screening with primers amplifying the sgRNA-1 target
region and the internal region of the Cre cassette (Fig. 2b, e).
Second PCR and sequencing analysis using primer sets
amplifying the 5′ genome-donor boundary and donor 3′
genome boundary (Supplementary Table 2) indicated indel
mutations at the sgRNA-2 targeting intron region and KI of
the P2A-Cre cassette before the terminal codon of Pvalb (#2)
and Th genes (#4 and #5). However, #3 rat carried the P2ACre cassette followed by the 3′ HA and the vector sequences,which indicates that the whole donor vector sequence was
integrated via the NHEJ repair pathway alone (Fig. 3).

Discussion
In this study, we report a novel CRISPR-mediated genome
engineering method, Combi-CRISPR, which combines the
NHEJ and HDR repair pathways for the efficient and precise
KI of a few kbp gene cassette in mice and rats. It is necessary to prepare a donor dsDNA with hundreds to 1 kbps
HAs and to design the first sgRNA (sgRNA-1) at the target
site where the KI cassette should be integrated in the same
way as for the conventional HDR-dependent KI method. It
is also necessary to design another sgRNA (sgRNA-2) targeting an arbitrary region close to the first targeting site andwithin the HA region where the incidence of CRISPR-mediated mutations is minimized, although indels in intronic or
3′UTR regions might have serious consequences. Similarly,
the main limit of our technique is that it is only applicable to a subset of KI strategies such as Tagging and cannot
be used for other strategies, such as humanizing an animal
gene, which cannot accommodate indels. Using this CombiCRISPR method, we demonstrated efficient KIs using various types of donor cassettes, such as EGFP, mCherry, Cre,and genes of interest, for seven targeting loci in mice andtwo in rats. A similar approach was recently reported as
HMEJ, which induced efficient recombination between
two DSBs in the genomic region and the homology armsof the dsDNA donor, although the HMEJ-mediated repair
mechanism remains unknown (Yao et al. 2017). Our PCR
and sequencing analysis on the KI founders always indicatedthat indel mutations via NHEJ repair were present at thesgRNA-2 target site. In contrast, the precise KI of the donor
cassettes without any small indel mutations via HDR weredetected at the sgRNA-1 target site in all KI animals, except
for one rat carrying a whole donor vector sequence, probably because plasmid integration occurred via NHEJ alone(Fig. 3). Therefore, we speculate that the first sgRNA-2/
Cas9 induces double-strand breaks (DSBs), and then, indel
mutations are induced via dominant NHEJ in zygotes at thefirst step. This first step may induce the assembly of several
factors associated with the DSB repair pathway, which may
then induce the efficient repair of the other DSB via HDR
between the genomic target region and the homologous armof the plasmid donor vector (Fig. 3).
There are several KI methods using the CRISPR-Cas9
system in mice and rats (Table 2). To exchange a single point
mutation or introduce small indels, ssODNs are widely used
as a donor template with CRISPR-Cas9. When sequences
longer than 50 bp are to be integrated, the lssDNA canalso be used in zygotes. Efficient KIs of simple Cre cassette sequences or flanked two loxP sites were previously
reported by Easi-CRISPR in mice (Quadros et al. 2017) andby CLICK in rats (Miyasaka et al. 2018). The advantage
of using ssDNA as a donor template is that electroporation
can be used. However, both short and long ssDNA methods
have size limitations for KIs, less than 100 bp and 2 kbp inzygotes, respectively. For KIs of longer cassette sequences, aconventional CRISPR-mediated KI method via HDR using a
dsDNA donor template is available, although its efficiency islow. Several other KI methods using dsDNAs as donor templates were reported for cultured cells and mice (Auer et al.
2014; Gu et al. 2018; He et al. 2016; Maresca et al. 2013;
Nakade et al. 2014; Suzuki et al. 2016, 2019) (Table 2).
ObLiGaRe, HITI, PITCH, HMEJ, and SATI are useful for
KI in in vitro cultures; however, these technologies have notbeen examined thoroughly in mouse and rat zygotes. TildCRISPR, based on an HMEJ strategy, was recently reported
using linear dsDNA as a donor template (Yao et al. 2018).
The Combi-CRISPR method uses circular dsDNA, althoughboth technologies use sgRNA to cut the targeted genome
sequences and the homology arm of the dsDNA, whichmight increase the recombination efficiency via HMEJ (Yaoet al. 2018). 2C-HR is a unique, highly efficient, and usefultechnology for gene KI in zygotes, except for the technicaldifficulties for general researchers and technicians (Gu et al.
2018). Our Combi-CRISPR method provides an efficient and13

Human Genetics

precise KI strategy in mouse and rat zygotes, which is suitable for projects that can accommodate indels in intronic orotherwise dispensable regions.

13

In this study, Combi-CRISPR provided efficient KIs
of approximately 10–33% in zygotes. However, there aresome disadvantages compared with other KI methods.

Human Genetics◂Fig. 2  Knock-in rats generated by injection of two sgRNAs, Cas9,

and a donor dsDNA in rat zygotes. a Methods to integrate the P2ACre cassette at the terminal codon of the Pvalb gene with lssDNA(above) or dsDNA (bottom). Microinjection of two sgRNA, Cas9,
and dsDNA provided a KI rat (#1) carrying precise KIs of the Cre
cassette at the sgRNA-1 targeting site and a 1 bp deletion mutation
at the sgRNA-2 targeting site. b, e Comparison of three methods
using dsDNA with single sgRNA-1 (HR), lssDNA with sgRNA-1(lssDNA), or dsDNA with two sgRNAs (Combi-CRISPR) for KIs in
rat zygotes. c, f PCR analysis using primer sets amplifying the internal region of the Cre cassette (first screening) or for 5′ genome-donor
boundary (Upstream) and donor-3′ genome boundary (Downstream insecond screening) in delivered rat pups (#1–2 for c and #1–5 for f).
M: 100 bp DNA ladder marker. d Methods to integrate the P2A-Cre
cassette at the terminal codon of the Th gene with lssDNA (above) or
dsDNA (bottom). Microinjection of two sgRNA, Cas9, and dsDNA
provided two KI rats (#4, 5) carrying precise KIs of the Cre cassetteat the sgRNA-1 targeting site and insertion or deletion mutations at
the sgRNA-2 targeting site. Integration of the P2A-Cre cassette withthe 3′ HA, and confirmation of the vector sequences by PCR andsequencing analysis in #3 rat

Combi-CRISPR generally induces indel mutations within
one homology arm because of the NHEJ repair pathway.
Therefore, there is a risk of affecting endogenous genes ortransgene expression by these indels even if these mutations are controllable in the intron. There is also a risk forthe random integration of dsDNA similar to transgenic
methods using linear dsDNA. In the F0 founders which we
tested, random insertions or complex rearrangements wereobserved among the Founders (such as Kcnab1 founders #3
and 4, Mc4r founder #7). Random integration might occur
anyway when dsDNA donors are used. In our case, cutting
circular plasmids by Cas9 inside cells might reduce random
integration and the integration of multiple copies of plasmids compared with other methods using linearized dsDNA.
NHEJ-mediated mutations or DNA insertions at off-target
sites may also eventually occur. A more comprehensive analysis (that is whole-genome sequencing) is required to assesson- and off-target events. Further backcrossing to wild-type
animals might segregate such integrations.
In conclusion, the Combi-CRISPR method is less timeconsuming, easier to prepare, and highly efficient for thegeneration of KI mice and rats for our tested genes. Donor
vector dsDNA, Cas9 protein, and two synthetic sgRNAs canalso easily be purchased from custom-order companies.

Methods
Animals and zygotes
Iar:Wistar-Imamichi pseudopregnant female rats and
Iar:Long-Evans rats (8–10 weeks old) were sourced fromJapan SLC, Inc (Hamamatsu, Japan). Iar:Long-Evans cryopreserved zygotes were obtained from the ARK resource(Kumamoto, Japan). Jcl:ICR pseudopregnant female miceand C57BL/6JJcl cryopreserved zygotes were purchasedfrom CLEA Japan Inc (Tokyo, Japan). All animals werehoused and maintained under conditions of 50% humidity
and a 12:12-h light:dark cycle. They were fed a standard
pellet diet (MF, Oriental Yeast Co., Tokyo, Japan) and tap
water ad libitum. The Osaka University Animal ExperimentCommittee approved all animal experiments.

Preparation of Cas9, sgRNAs, and plasmids
Production and purification of Cas9 mRNA were performed
as described previously (Yoshimi et al. 2014, 2016). Cas9
protein was obtained from IDT (Alt-R S.p. Cas9 Nuclease
V3, Integrated DNA Technologies, IA, USA). sgRNAs were
designed using an online program (https​://crisp​or.tefor​.net/)
to predict unique target sites throughout the mouse and rat
genome. Single-guide RNAs were transcribed in vitro fromsynthetic double-strand DNAs obtained from IDT or Thermo
Fisher Scientific using a MEGAshortscript T7 Transcription
Kit (Thermo Fisher Scientific, MA, USA). Specific crRNAs
were purchased from IDT (Alt-R CRISPR-Cas9 crRNA)
and were assembled with a tracrRNA (Alt-R CRISPR-Cas9
tracrRNA) before use according to the instructions of the
manufacturer. Several plasmids used as knock-in donors
were purchased from Thermo Fisher Scientific (GeneArt
Gene Synthesis). In accordance with the conventional
methods, all plasmids were transformed into Escherichia
coli and extracted with NucleoSpin Plasmid Transfection
grade (MACHEREY–NAGEL Gmbh & Co. KG, Germany).

Microinjections into mouse and rat embryos
Pronuclear-stage mouse embryos were prepared by thawing frozen embryos in KSOM medium (ARK Resource,
Kumamoto, Japan) and incubating them for 2–3 h before
microinjection. Pronuclear-stage rat embryos were preparedby thawing frozen embryos 2–3 h before microinjection orcollecting fresh embryos from naturally mated female rats.
Female rats were superovulated by the administration of
150 U/kg of PMSG followed 46–47 h later by 75 U/kg of
HCG and mating 1:1 with males. The next day, all females
exhibiting copulation plugs were sacrificed and pronuclear
embryos were collected from oviducts and maintained under
5% ­CO2 at 37 °C for 2–3 h. All rat embryos were cultured
in Rat KSOM medium (ARK Resource). A solution containing 20 ng/µl Cas9 mRNA, 25 ng/µl sgRNA-1, 25 ng/µl
sgRNA-2, and 1–3 ng/µl donor plasmid were microinjected
into male pronuclei and cytoplasm of mouse embryos using
a micromanipulator (Narishige, Tokyo, Japan). Likewise,
100 ng/µl Cas9 protein, 25 ng/µl sgRNA-1, 25 ng/µl sgRNA2, and 1 ng/µl donor plasmid were microinjected into malepronuclei and cytoplasm of rat embryos. All surviving

13

Human Genetics
Fig. 3  Schematic representation of precise and efficient
knock-ins by Combi-CRISPR. A
dsDNA donor vector was usedwith Cas9 and two sgRNAs,
one designed to cut the targeted
genome sequences (sgRNA-2)
and the other to cut both theflanked genomic region and one
homology arm of the dsDNA
plasmid (sgRNA-1 targeting).
The NHEJ repair pathway
dominantly induces indel mutations (purple) at the sgRNA-2
targeting site. Thereafter, theHDR pathway integrates a KI
cassette (red) without any mutation at the sgRNA-1 targetingsite. In some cases, the whole
donor vector was integrated at
the sgRNA-2 targeting site via
NHEJ (black)embryos were transferred on the same day or next day into
the oviducts of pseudopregnant surrogate mothers anesthetized with isoflurane (DS Pharma Animal Health Co., Ltd.,
Osaka, Japan).

Genotyping analysis
For PCR and sequence analysis, genomic DNA wasextracted from a tail biopsy with the KAPA Express Extract

13

DNA Extraction Kit (Kapa Biosystems, London, UK). The
sgRNA targeted region, 5′ genome-donor boundary, insideof knock-in donor, and donor-3′ genome boundary were
PCR amplified. These PCR amplicons were then directly
sequenced using the BigDye Terminator v3.1 cycle sequencing mix and the standard protocol for an Applied Biosystems
3130 DNA Sequencer (Thermo Fisher Scientific). All primer
sets used for genotyping analysis are shown in Supplemental
Table 2.

Human Genetics
Table 2  Various knock-in methods in mouse zygotes with genome editing technology
MethodRepairDonor

Vector cut Homology
arms

Efficiency Accuracy Size (bp) References

ConventionalHR

Homologous
recombination (HR)

dsDNA

−

200–1 K

Low

High

1–10 K

ssODN

Homology
directed
repair(HDR)
Non-homologous end
joining(NHEJ)

ssDNA

−

40–80

High

High

1–100

dsDNA

+

–

–

Low

1–10 K

dsDNA

+

20

Low

High

1–10 K

dsDNA

+

20

Low

Low

1–10 K

Suzuki et al.
(2016)

dsDNA+

200–1 K

High

High

1–10 K

Suzuki et al.
(2019)

dsDNA

+

–

Low

Low

1–200 K Yoshimi et al.
(2016)

dsDNA−

200–1 K

High

High

1–10 K

Gu et al. (2018)

dsDNA

+

200–1 K

Low

Low

1–10 K

dsDNA

linear

200–1 KHigh

Low

1–10 K

Yao et al.
(2017), Zhang
et al. (2017)
Yao et al. (2018)

long ssDNA −50–300

High

High

1–2 K

Quadros et al.
(2017)

long ssDNA −50–300

High

High

1–2 K

Miyasaka et al.
(2018)ObLiGaRe

Obligate ligation-gatedrecombination

PITCh

MicrohoPrecise
mologyintegration
mediated
into target
end-joining
chromosome(MMEJ)
NHEJ
Homologyindependent
targeted
integration
NHEJ and HR
intercellularlinearized
singlehomology
arm donormediatedintrontargeting
integration
NHEJ?
Two-hit andtwo oligos
with a targeting plasmid
HDR
Two-cell
homologous
recombination
NHEJ? or
HomologyHR?
mediatedend joining
Targeted inte- NHEJ? orHR?
gration withlinearized
dsDNACRISPR
HDR
Efficientadditionswith ssDNA
insertsCRISPRCRISPR with HDR
lssDNA
inducing
conditional
knockout
alleles

HITI

SATI

2H2OP

2C-HR

HMEJ
Tild-CRISPR

Easi-CRISPR

CLICK

Lin et al. (2018),
Ma et al.
(2014), Yanget al. (2013)
Wang et al.
(2013),
Yoshimi et al.
(2014)
Auer et al.
(2014), Heet al. (2016),
Maresca et al.
(2013)
Nakade et al.
(2014)

13

Human Genetics
Table 2  (continued)
MethodRepairDonor

CombiCRISPRNHEJ and HR dsDNA
Combinationof NHEJ
and HR withCRISPR

Vector cut Homology
arms

Efficiency Accuracy Size (bp) References

+

High

Acknowledgements We would like to thank Kanako Shimizu at Osaka
University for experimental assistance. We would also like to thankEdanz Group (www.edanz​editi​ng.com/ac) for editing a draft of thismanuscript.
Author contributions TM conceived the study, analyzed the data,
and wrote the paper. KY, YK, YU, and AT performed MI and EL of
CRISPR-Cas9 and donor templates into mouse embryos. YM and KH
performed MI of CRISPR-Cas9 and donor templates into rat embryos.
YO, MY, MS, and KN performed the mouse experiments, PCR, andsequence analyses. NM and KK performed the rat experiments, PCR,
and sequence analyses. All authors have read and approved the manuscript before submission.
Funding This study was supported in part by JSPS KAKENHI from
the Ministry of Education, Culture, Sports, Science and Technologyof Japan (MEXT) (T.M.).
Availability of data and materials All data generated or analyzed during
this study are included in this published article and its supplementary
information files.

Compliance with ethical standards
Conflict of interest The authors have declared that no conflict of interest exists.
Ethics approval and consent to participate Animal care and experiments conformed to the Guidelines for Animal Experiments of Osaka
University, and were approved by the Animal Research Committee of
Osaka University.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as longas you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article areincluded in the article’s Creative Commons licence, unless indicatedotherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view acopy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/.

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