One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering

Article (PDF Available)inCell 154(6) · August 2013with176 Reads
DOI: 10.1016/j.cell.2013.08.022 · Source: PubMed
Abstract
The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.
One-Step Generation of Mice Carrying
Reporter and Conditional Alleles by
CRISPR/Cas-Mediated Genome Engineering
Hui Yang,
1,4
Haoyi Wang,
1,4
Chikdu S. Shivalila,
1,2,4
Albert W. Cheng,
1,3
Linyu Shi,
1
and Rudolf Jaenisch
1,3,
*
1
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA
2
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
3
Computational and Systems Biology Program, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
4
These authors contributed equally to this work
*Correspondence: jaenisch@wi.mit.edu
http://dx.doi.org/10.1016/j.cell.2013.08.022
SUMMARY
The type II bacterial CRISPR/Cas system is a novel
genome-engineering technology with the ease of
multiplexed gene targeting. Here, we created
reporter and conditional mutant mice by coinjection
of zygotes with Cas9 mRNA and different guide
RNAs (sgRNAs) as well as DNA vectors of different
sizes. Using this one-step procedure we generated
mice carrying a tag or a fluorescent reporter
construct in the Nanog, the Sox2, and the Oct4
gene as well as Mecp2 conditional mutant mice. In
addition, using sg RNAs targeting two separate sites
in the Mecp2 gene, we produced mice harboring the
predicted deletions of about 700 bps. Finally, we
analyzed potential off-targets of five sgRNAs in
gene-modified mice and ESC lines and identified
off-target mutations in only rare instances.
INTRODUCTION
Mice with specific gene modification are valuable tools for
studying development and disease. Traditional gene targeting
in embryonic stem (ES) cells, although suitable for generating
sophisticated genetic modifications in endogenous genes, is
complex and time-consuming (Capecchi, 2005). The production
of genetically modified mice and rats has been greatly acceler-
ated by novel approaches using direct injection of DNA or
mRNA of site-specific nucleases into the one-cell-stage embryo,
generating DNA double-strand breaks (DSB) at specified se-
quences leading to targeted mutations (Carbery et al., 2010;
Geurts et al., 2009; Shen et al., 2013; Sung et al., 2013; Tesson
et al., 2011; Wang et al., 2013). Coinjection of a single-stranded
or double-stranded DNA template containing homology to the
sequences flanking the DSB can produce mutant alleles with
precise point mutations or DNA inserts (Brown et al., 2013; Cui
et al., 2011; Meyer et al., 2010; Wang et al., 2013; Wefers
et al., 2013). Recently, pronuclear injection of two pairs of
ZFNs and two double-stranded donor vectors into rat fertilized
eggs produced rat containing loxP-flanked (floxed) alleles
(Brown et al., 2013). However, the complex and time-consuming
design and generation of ZFNs and double-stranded donor vec-
tors limit the application of this method.
CRISPR (clustered regularly interspaced short palindromic
repeat) and Cas (CRISPR-associated) proteins function as the
RNA-based adaptive immune system in bacteria and archaea
(Horvath and Barrangou, 2010; Wiedenheft et al., 2012). The
type II bacterial CRISPR/Cas system has been demonstrated
as an efficient gene-targeting technology that facilitates multi-
plexed gene targeting (Cong et al., 2013; Wang et al., 2013).
Because the binding of Cas9 is guided by the simple base-pair
complementarities between the engineered single-guide RNA
(sgRNA) and a target genomic DNA sequence, it is possible to
direct Cas9 to any genomic locus by providing the engineered
sgRNA (Cho et al., 2013; Cong et al., 2013; Gilbert et al., 2013;
Hwang et al., 2013; Jinek et al., 2012; Jinek et al., 2013; Mali
et al., 2013b; Qi et al., 2013; Wang et al., 2013).
Previously, we used the type II bacterial CRISPR/Cas system
as an efficient tool to generate mice carrying mutations in multi-
ple genes in one step (Wang et al., 2013). However, this study left
a number of issues unresolved. For example, neither the effi-
ciency of using the CRISPR/Cas gene-editing approach for the
insertion of DNA constructs into endogenous genes nor its utility
to create conditional mutant mice was clarified. Here, we report
the one-step generation of mice carrying reporter constructs in
three different genes as well as the derivation of conditional
mutant mice. In addition, we performed an extensive off-target
cleavage analysis and show that off-target mutations are rare
in targeted mice and ES cells derived from CRISPR/Cas zygote
injection.
RESULTS
Targeted Insertion of Short DNA Fragments
In previous work, we introduced precise base pair mutations into
the Tet1 and Tet2 genes through homology directed repair
(HDR)-mediated genome editing following coinjection of single-
stranded mutant DNA oligos, sgRNAs, and Cas9 mRNA (Wang
et al., 2013). To test whether a larger DNA construct could be in-
serted at the same DSBs at Tet1 exon 4 and Tet2 exon 3, we
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designed oligos containing the 34 bp loxP site and a 6 bp EcoRI
site flanked by 60 bps sequences on each side adjoining the
DSBs (Figure S1A available online). We coinjected Cas9
mRNA, sgRNAs, and single-stranded DNA oligos targeting
both Tet1 and Tet2 into zygotes. The restriction fragment length
polymorphism (RFLP) assay shown in Figure S1B identified 6 out
of 15 tested embryos carrying the loxP site at the Tet1 locus, 8
carrying the loxP site at the Tet2 locus, and 3 had at least one
allele of each gene correctly modified. The correct integration
of loxP sites was confirmed by sequencing (Figure S1C). These
results demonstrate that HDR-mediated repair can introduce
targeted integration of 40 bp DNA elements efficiently through
CRISPR/Cas-mediated genome editing (summarized in Table 1).
Mice with Reporters in the Endogenous Nanog, Sox2,
and Oct4 Genes
Because the study of many genes and their protein products are
limited by the availability of high-quality antibodies, we explored
the potential of fusing a short epitope tag to an endogenous
gene. We designed a sgRNA targeting the stop codon of Sox2
and a corresponding oligo to fuse the 42 bp V5 tag into the last
codon (Figure 1A). After injection of the sgRNA, Cas9 mRNA,
and the oligo into zygotes, in vitro differentiated blastocysts
were explanted into culture to derive ES cells. PCR genotyping
and sequencing identified 7 out of 16 ES cell lines carrying a
correctly targeted insert (Figures 1B and 1C). Western blot anal-
ysis revealed a protein band at the predicted size using V5 anti-
body in targeted ES cells but not in the control cells (Figure 1D).
As expected from a correctly targeted and functional allele, Sox2
expression was seen in targeted blastocysts and ES cells using
V5 antibody (Figures 1E and 1F). Twelve of 35 E13.5 embryos
and live-born mice derived from injected zygotes carried the
V5 tag correctly targeted into the Sox2 gene as indicated by
PCR genotyping and sequencing (data not shown, Table 1).
To assess whether a marker transgene could be inserted into
an endogenous locus, we coinjected Cas9 mRNA, sgRNA, and a
double-stranded donor vector that was designed to fuse a p2A-
mCherry reporter with the last codon of the Nanog gene ( Fig-
ure 2A). A circular donor vector was used to minimize random
integrations. To assess toxicity and to optimize the concentra-
tion of donor DNA, we microinjected different amounts of
Nanog-2A-mCherry vector. Injection with a high concentration
of donor DNA (500 ng/ml) yielded mCherry-positive embryos
with high efficiency, with most blastocysts being retarded,
whereas injection with a lower donor DNA concentration
(10 ng/ml) yielded mostly healthy blastocysts, most of which
were mCherry-negative. When 200 ng/ml donor DNA was used,
75% (936/1,262) of the injected zygotes developed to blasto-
cysts, 9% (86/936) of which were mCherry-positive (Figure 2C;
Table S1). mCherry was mainly expressed in the inner cell
mass (ICM), consistent with targeted integration of the mCherry
transgene into the Nanog gene. We derived six ES cell lines from
mCherry-positive blastocysts, four of which uniformly expressed
mCherry with the signal disappearing upon cellular differentia-
tion (Figure 2C). The other two lines showed variegated mCherry
expression, with some colonies being mCherry positive and
others negative (Figure S2A, Table S2) consistent with mosaic
donor embryos, which would be expected if transgene insertion
occurred later than the zygote stage, as has been previously
observed with ZNF and TALEN-mediated targeting (Brown
et al., 2013; Cui et al., 2011; Wefers et al., 2013). Correct trans-
gene integration in ES cell lines was confirmed by Southern
blot analysis (Figure 2B). We also generated mice from injected
zygotes. Southern blot analysis (Figures S2B and S2C) revealed
that 7 out of 86 E13.5 embryos and live-born mice carried the
mCherry transgene in the Nanog locus. One targeted mouse
was mosaic (Table S2), because the intensity of targeted allele
was lower than the wild-type allele (Figure S2B, #6). Two of the
mice carried an additional randomly integrated transgene (Fig-
ure S2C, #3). As summarized in Tables 1 and S1, the efficiency
of targeted insertion of the transgene was about 10% in blasto-
cysts and mice derived from injected zygotes.
Finally, we designed sgRNA targeting the Oct4 3
0
UTR, which
was coinjected with a published donor vector designed to inte-
grate the 3 kb transgene cassette (IRES-eGFP-loxP-Neo-loxP;
Figure 2D) at the 3
0
end of the Oct4 gene (Lengner et al., 2007).
Blastocysts were derived from injected zygotes, inspected for
GFP expression, and explanted to derive ES cells. About 20%
(47/254) of the blastocysts displayed uniform GFP expression
in the ICM region. Three of nine derived ES cell lines expressed
GFP (Figure 2E), including one showed mosaic expression (Table
S2). Three out of ten live-born mice contained the targeted allele
(Table 1). Correct targeting in mice and ES cell lines was
confirmed by Southern blot analysis (Figure 2F).
Table 1. Mice with Reporters in the Endogenous Genes
Donor
Blastocysts/Injected
Zygotes
Targeted
Blastocysts/Total
Targeted
ESCs/Total
Transferred embryos
(recipients)
Knockin pre- and
postnatal mice/Total
Tet1-loxP + Tet2-loxP 65/89 Tet1-loxP 6/15 N/A N/A N/A
Tet2-loxP 8/15
Both 3/15
Sox2-V5 414/498 ND 7/16 200(10) 12/35
Nanog-mCherry 936/1262 86/936 ND 415 (21) 7/86
Oct4-GFP 254/345 47/254 3/9 100(4) 3/10
Cas9 mRNA, sgRNAs targeting Tet1, Tet2, Sox2, Nanog, or Oct4, and single-stranded DNA oligos or double-stranded donor vectors were injected into
fertilized eggs. Targeted blastocysts were identified by RFLP or fluorescence of reporters. The blastocysts derived from injected embryos were derived
ES cell lines or transplanted into foster mothers and E13.5 embryos and postnatal mice were obtained and genotyped.
ND, not determined.
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Figure 1. One-Step Generation of the Sox2-V5 Allele
(A) Schematic of the Cas9/sgRNA /oligo-targeting site at the Sox2 stop codon. The sgRNA coding sequence is underlined, capitalized, and labeled in red. The
protospacer-adjacent motif (PAM) sequence is labeled in green. The stop codon of Sox2 is labeled in orange. The oligo contained 60 bp homologies on both sides
flanking the DSB. In the oligo donor sequence, the V5 tag sequence is labeled as a green box. PCR primers (SF, V5F, and SR) used for PCR genotyping are shown
as red arrowheads.
(B) Top: PCR genotyping using primers V5F and SR produced bands with correct size in targeted ES samples T1 to T5, but not in WT sample. Bottom: PCR
genotyping using primers SF and SR produced slightly larger products, indicating the 42 bp V5 tag sequence was integrated. T1 only contain larger product,
suggesting either both alleles were targeted, or one allele failed to amplify.
(C) PCR products using primers SF and SR were cloned into plasmid and sequenced. Sequence across the targeting region confirmed correct fusion of V5 tag to
the last codon of Sox2.
(D) Western blot analysis identified Sox2-V5 protein using V5 antibody in ES cells containing Sox2-V5 allele. Beta-actin was shown as the loading control.
Because beta-actin and Sox2-V5 run at the same size, the samples for the V5 signal and beta-actin were run in parallel on the same gel.
(E) Immunostaining of targeted blastocyst using V5 antibody showed signal in ICM. Scale bar, 50 mm.
(F) Immunostaining of targeted ES cells using V5 antibody showed uniform Sox2 expression. Scale bar, 100 mm.
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(legend on next page)
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Text
Conventionally, transgenic mice are generated by pronuclear
instead of cytoplasmic injection of DNA. To optimize the
generation of CRISPR/Cas9-targeted embryos, we compared
different concentrations of RNA and the Nanog-mCherry or the
Oct4-GFP DNA vectors as well as three different delivery modes:
(1) simultaneous injection of all constructs into the cytoplasm, (2)
simultaneous injection of the RNA and the DNA into the pronu-
cleus, and (3) injection of Cas9/sgRNA into the cytoplasm fol-
lowed 2 hr later by pronuclear injection of the DNA vector. Table
S1 shows that simultaneous injection of all constructs into the
cytoplasm at a concentration of 100 ng/ml Cas9 RNA, 50 ng/ml
of sgRNA and 200 ng/ml of vector DNA was optimal, resulting
in 9% (86/936) to 19% (47/254) of targeted blastocysts. Similarly,
the simultaneous injection of 5 ng/ml Cas9 RNA, 2.5 ng/ml of
sgRNA, and 10 ng/ml of DNA vector into the pronucleus yielded
between 9% (7/75) and 18% (13/72) targeted blastocysts. In
contrast, the two-step procedure with Cas9 and sgRNA simulta-
neous injected into the cytoplasm followed 2 hr later by pronu-
clear injection of different concentrations of DNA vector yielded
no or at most 3% (1/34) positive blastocysts. Thus, our results
suggest that simultaneous injection of RNA and DNA into the
cytoplasm or nucleus is the most efficient procedure to achieve
targeted insertion.
Conditional Mecp2 Mutant Mice
We investigated whether conditional mutant mice can be gener-
ated in one step by insertion of two loxP sites into the same allele
of the Mecp2 gene. To derive conditional mutant mice similar to
those previously described using traditional homologous recom-
bination methods in ES cells (Chen et al., 2001), we designed two
sgRNAs targeting Mecp2 intron 2 (L1, L2) and two sgRNAs
targeting intron 3 (R1, R2), as well as the corresponding loxP
site oligos with 60 bp homology to sequences on each side sur-
rounding each sgRNA-mediated DSB (Figure S3A). To facilitate
detection of correct insertions, the oligos targeting intron 2
were engineered to contain a NheI restriction site and the oligos
targeting intron 3 to contain an EcoRI site in addition to the loxP
sequences (Figures 3A and S3A). To determine the efficiency of
single loxP site integration at the Mecp2 locus, we injected Cas9
mRNA and each single sgRNA and corresponding oligo into
zygotes, which were cultured to the blastocyst stage and geno-
typed by the RFLP assay. As shown in Figure S3B, the L2 and R1
sgRNAs were more efficient in integrating the oligos with four out
of eight embryos carrying the L2 oligo and two out of six embryos
carrying the R1 oligo. Therefore, L2 and R1 sgRNAs and the
corresponding oligos were chosen for the generation of a floxed
allele (Figure 3A).
A total of 98 E13.5 embryos and mice were generated from
zygotes injected with Cas9 mRNA, sgRNAs, and DNA oligos tar-
geting the L2 and R1 sites. Genomic DNA was digested with both
NheI and EcoRI, and analyzed by Southern blot using exon 3 and
exon 4 probes (Figures 3A and 3B). The L2 and R1 oligos con-
tained, in addition to the loxP site, different restriction sites
(NheI or EcoRI). Thus, single loxP site integration at L2 or R1
will produce either a 3.9 kb or a 2 kb band, respectively, when
hybridized with the exon3 probe (Figures 3A and B). We found
that about 50% (45/98) of the embryos and mice carried a loxP
site at the L2 site and about 25% (25/98) at the R1 site. Impor-
tantly, integration of both loxP sites on the same DNA molecule,
generating a floxed allele, produces a 700 bp band as detected
by exon 3 probe hybridization (Figures 3A and 3B). RFLP anal-
ysis, sequencing (Figures S4A and S4B) and Southern blot
analysis (Figure 3 B) showed that 16 out of the 98 mice tested
contained two loxP sites flanking exon 3 on the same allele.
Table 2 summarizes the frequency of all alleles and shows that
the overall insertion frequency of an L2 or R1 insertion was
slightly higher in females (21/38) than in males (28/60) consistent
with the higher copy number of the X-linked Mecp2 gene in
females. To confirm that the floxed allele was functional, we
used genomic DNA for in vitro Cre-mediated recombination.
Upon Cre treatment, both the deletion and circular products
were detected by PCR in targeted mice, but not in DNA from
wild-type mice (Figure 3C). The PCR products were sequenced
and confirmed the precise Cre-loxP-mediated recombination
(Figure S4C).
We noticed that some pups carried large deletions but no loxP
insertions, raising the possibility that two cleavage events may
generate defined deletions. To confirm this notion, we coinjected
Cas9 mRNA, Mecp2-L2, and R1 sgRNAs but without oligos.
PCR genotyping and sequencing (Figures 3D and 3E) revealed
that 8 out of 23 mice carried deletions of about 700 bp spanning
the L2 and R1 sites removing exon 3. This was confirmed by
Figure 2. One-Step Generation of an Endogenous Reporter Allele
(A) Schematic overview of strategy to generate a Nanog-mCherry knockin allele. The sgRNA coding sequence is underlined, capitalized, and labeled in red. The
protospacer-adjacent motif (PAM) sequence is labeled in green. The stop codon of Nanog is labeled in orange. The homologous arms of the donor vector are
indicated as HA-L (2 kb) and HA-R (3 kb). The restriction enzyme used for Southern blot analysis is shown, and the Southern blot probes are shown as red boxes.
(B) Southern analysis of Nanog-mCherry targeted allele. NcoI-digested genomic DNA was hybridized with 3
0
external probe. Expected fragment size: WT (wild-
type) = 11.5 kb, T (targeted) = 5.6 kb. The blot was then stripped and hybridized with mCherry internal probe. Expected fragment size: WT = N/A, T = 6.6 kb.
(C) Nanog-mCherry targeted blastocysts showed expression in ICM. Mouse ES cell lines derived from targeted blastocysts remain mCherry positive, and the
mCherry expression disappear upon differentiation. Scale bar, 100 mm.
(D) Schematic overview of strategy to generate an Oct4-eGFP knockin allele. The sgRNA coding sequence is underlined, capitalized, and labeled in red. The PAM
sequence is labeled in green. The homologous arms of the donor vector are indicated as HA-L (4.5 kb) and HA-R (2 kb). The IRES-eGFP transgene is indicated as
a green box, and the PGK-Neo cassette is indicated as a gray box. The restriction enzyme used for Southern blot analysis is shown, and the Southern blot probes
are shown as red boxes.
(E) Oct4-eGFP targeted blastocysts showed expression in ICM. Scale bar, 50 mm. Mouse ES cell lines derived from targeted blastocysts remain GFP positive.
Scale bar, 100 mm.
(F) Southern analysis of Oct4-eGFP targeted allele. Southern analysis of Oct4-eGFP targeted allele. HindIII-digested genomic DNA was hybridized with 3
0
external
probe. Expected fragment size: WT = 9 kb, Targeted = 7.2 kb. The blot was then stripped and hybridized with eGFP internal probe. Expected fragment size: WT =
N/A, Targeted = 7.2 kb. See also Figure S2.
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Figure 3. One-Step Generation of a Mecp2 Floxed Allele
(A) Schematic of the Cas9/sgRNA/oligo targeting sites in Mecp2 intron 2 and intron 3. The sgRNA coding sequence is underlined, capitalized, and labeled in red.
The PAM sequence is labeled in green. In the oligo donor sequence, the loxP site is indicated as an orange box, and the restriction site sequences are in bold and
capitalized. The oligo contained 60 bp homologies on both sides flanking the DSB. Restriction enzymes used for RFLP and Southern blot analysis are shown, and
the Southern blot probes are shown as red boxes.
(B) Southern analysis of targeted alleles. Data of five mice are shown. EcoRI/NheI-digested genomic DNA was hybridized with the exon3 probe. Expected
fragment size: WT = 5.2 kb, 2loxP = 0.7 kb, L2-loxP = 3.9 kb, and R1-loxP = 2 kb. The blot was then stripped and hybridized with the exon 4 probe. Expected
fragment size: WT = 5.2 kb, 2loxP = 3.2 kb, L2-loxP = 3.9 kb, and R1-loxP = 3.2 kb. The sequence of the floxed allele is shown in Figure S4B.
(legend continued on next page)
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Southern analysis (data not shown). Because DNA breaks are
repaired through the nonhomologous end joining (NHEJ)
pathway, the ends of the breaks are different in different deletion
alleles (Figure 3E).
Mosaicism
As mentioned above, we noted that some animals were mosaic
for the targeted insertion. We decided to characterize the fre-
quency of mosaicism in Mecp2-targeted mice by Southern blot
analysis. Because Mecp2 is an X-linked gene, in males more
than one allele and in females more than two different alleles sug-
gest mosaicism, which would be expected if integration occurred
after the zygote stage. For example, as shown in Figure 3B,
female mouse #2 contained three different alleles (one WT allele,
one floxed allele, and one L2-loxP allele), and female mouse #4
contained four different alleles (one WT allele, one floxed allele,
one L2-loxP allele, and one R1-loxP allele). Male mouse #5 con-
tained two different alleles, with each allele carrying a single loxP
site (Figure 3B). We identified eight mosaics out of 16 mice con-
taining a Mecp2 floxed allele. The frequency of mosaicism among
49 embryos and mice containing loxP site was about 40% (20/49)
(Table S2). Because Southern blot analysis cannot detect
small in-del mutations caused by NHEJ repair, it is possible
that this underestimates the overall mosaicism frequency.
Off-Target Analysis
Two recent studies identified a high level of off-target cleavage in
human cell lines using the CRISPR/Cas system, with Cas9-
targeting specificity being shown to tolerate small numbers of
mismatches between sgRNA and target DNA in a sequence
and position-dependent manner (Fu et al., 2013; Hsu et al.,
2013). Similarly, using a transcription-based method, Mali et al.
(2013a) reported that Cas9/sgRNA binding could tolerate up to
three mismatches.
We characterized potential off-target (OT) mutations in mice
and ES cell lines derived from zygotes injected with Cas9 and
sgRNAs targeting the Sox2, the Nanog, the Oct4, and the
Mecp2 gene. We identified all genomic loci containing up to
three or four base pair mismatches compared to the 20 bp
sgRNA coding sequence (Table S3). We amplified all 13 potential
OT sites of Sox2 sgRNA in six mice and four ES cell lines carrying
the Sox2-V5 allele and tested for potential off target mutations
using the Surveyor assay. No mutation was detected in any
locus. When nine Nanog sgRNA potential OT sites (including
seven genomic loci containing four base pair mismatches in
the PAM distal region) were tested in five correctly targeted
mice and four targeted ES cell lines, mutations were found in
seven samples at OT1 (Table S3). Since Nanog OT1 has only
one base pair difference at the very 5
0
end of the sgRNA (position
20, numbered 1–20 in the 3
0
to 5
0
direction of sgRNA target site),
it may not be surprising to find such a high frequency of muta-
tions at this locus. In contrast, no off-target mutation was seen
in any other Nanog OTs, which contain three or four base pair dif-
ference. For Oct4, we tested all 11 OT sites containing up to
three base pair mismatches in three targeted mice and three
targeted ES cell lines. Mutations were found in four out of six
samples at OT1, which has only one base pair mismatch at
position 19, whereas no off-target mutation was identified in
any other Oct4 OTs, which contain two or three base pair mis-
matches (Table S3). Finally, four potential off-targets sites for
Mecp2 L2, and ten sites for Mecp2 R1 were analyzed in ten
mice carrying a Mecp2 floxed allele. Only one off-target mutation
was identified in one mouse at the Mecp2 R1 OT2 (Table S3). In
summary, we tested all potential off-target sites differing by up
to three or four base pairs in 35 mice or ES cell lines and only
identified mutations in one off-target site for Nanog (OT1 7/9
samples, one mismatch at position 20), Oct4 (OT1 4/6 samples,
1 mismatch at position 19), and Mecp2 (R1-OT2 1/10 samples,
(C) In vitro Cre-mediated recombination of the floxed Mecp2 allele. The genomic DNA of targeted mice #1 and #3 was incubated with Cre recombinase, and used
as PCR template. Primers DF and DR flanking the floxed allele produce shorter products upon Cre-dependent excision. Primers CF and CR detect the circular
molecule, which only form upon Cre-loxP recombination. The position of each primer is shown at the bottom cartoon. The deletion and circular PCR products
were sequenced and the sequences are shown in Figure S4C.
(D) Injection of Cas9 mRNA and both L2 and R1 sgRNA generated Mecp2 mutant allele with deletion of exon 3. PCR genotyping using primers DF and DR
identified defined deletion events in mice #1, #6, and #8 (indicated by stars).
(E) Sequences of three mutant alleles with exon 3 deletions in three mice. R2 and L1 sgRNA coding sequences were underlined, capitalized, and labeled in red.
The PAM sequence is labeled in green. See also Figures S3 and S4.
Table 2. Conditional Mecp2 Mutant Mice
Donor
Blastocyst/Injected
Zygotes
Transferred Embryos
(Recipients) Sex
Pre- and Postnatal Mice with loxP/Total
Total
a
L2
b
R1
c
Two loxP in
Two Alleles
Two loxP in
One Allele
Mecp2-L2 + Mecp2-R1 367/451 360(18) Male 28/60 26/60 12/60 2
d
/60 8/60
Female 21/38 19/38 13/38 3/38 8/38
Total 49/98 45/98 25/98 5/98 16/98
Cas9 mRNA, sgRNAs targeting Mecp2-L2 and Mecp2-R1, and single-stranded DNA oligos were injected into fertilized eggs. The blastocysts derived
from the injected embryos were transplanted into foster mothers and pre- and postnatal mice were genotyped.
a
Total mice containing loxP site integration in the genome.
b
Mice containing loxP site integra ted at L2 site.
c
Mice containing loxP site integra ted at R1 site.
d
These male mice were mosaic.
Cell 154, 1–10, September 12, 2013 ª2013 Elsevier Inc. 7
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ated Genome Engineering, Cell (2013), http://dx.doi.org/10.1016/j.cell.2013.08.022
two mismatches at positions 7 and 20). No off target mutation
was identified in any genomic locus containing three base pair
mismatches. Thus, although the off-target mutation rate was
lower than what had been observed in the previous studies using
cultured human cancer cell lines, our results are consistent with
the conclusion that two or more interspaced mismatches
dramatically reduce Cas9 cleavage (Fu et al., 2013; Hsu et al.,
2013).
DISCUSSION
In this study, we demonstrate that CRISPR/Cas technology can
be used for efficient one-step insertions of a short epitope or
longer fluorescent tags into precise genomic locations, which
will facilitate the generation of mice carrying reporters in endog-
enous genes. Mice and embryos carrying reporter constructs in
the Sox2, the Nanog and the Oct4 gene were derived from
zygotes injected with Cas9 mRNA, sgRNAs, and DNA oligos or
vectors encoding a tag or a fluorescent marker. Moreover,
microinjection of two Mecp2-specific sgRNAs, Cas9 mRNA,
and two different oligos encoding loxP sites into fertilized eggs
allowed for the one-step generation of conditional mutant
mice. In addition, we show that the introduction of two spaced
sgRNAs targeting the Mecp2 gene can produce mice carrying
defined deletions of about 700 bp. Though all RNA and DNA con-
structs were injected into the cytoplasm or nucleus of zygotes,
the gene modification events could happen at the one-cell stage
or later. Indeed, Southern analyses revealed mosaicism in 17%
(1/6) to 40% (20/49) of the targeted mice and ES cell lines indi-
cating that the insertion of the transgenes had occurred after
the zygote stage (Table S2).
Our previous experiments (Wang et al., 2013) demonstrated
an efficiency of CRISPR/Cas sgRNA-mediated cleavage that
was high enough to allow for the one-step production of engi-
neered mice up to 90% of which carried homozygous mutations
in two genes (four mutant alleles). The results reported here show
that the sgRNA-mediated DSBs occur at a significantly higher
frequency than insertion of exogenous DNA sequences. There-
fore, the allele not carrying the insert will likely be mutated as a
consequence of NHEJ-based gene disruption. Thus, the re-
porter allele would need to be segregated away from the mutant
allele in order to produce mice carrying a reporter as well as a
wild-type allele.
Two recent studies reported a high off-target mutation rate in
CRISPR/Cas9-transfected human cell lines (Fu et al., 2013; Hsu
et al., 2013). We analyzed the off-target rate for five different
sgRNAs and identified cleavage of Nanog OT1, Oct4 OT1, and
Mecp2 R1 OT2. Nanog OT1 has only one base pair difference
from the targeting sequence at the extreme 5
0
end (position
20), whereas Oct4 OT1 contains one base pair mismatch at
position 19; and Mecp2 R1 OT2 has one base pair mismatch
at position 20, and one mismatch at position 7. Thus, the only
off-target mutations in 2 bp mismatch targets were seen when
one of the mismatches was at the distal 5
0
end. This result is
consistent with previous findings that Cas9 can catalyze DNA
cleavage in the presence of single-base mismatches in the
PAM distal region (Cong et al., 2013; Hsu et al., 2013; Jiang
et al., 2013; Jinek et al., 2012). No mutations were detected in
42 potential OTs of Sox2, Nanog, Oct4, or Mecp2 containing 3
or 4 bp mismatches in a total of 35 mice and ES cell lines tested,
consistent with the observation that three or more interspaced
mismatches dramatically reduce Cas9 cleavage (Hsu et al.,
2013). Thus, for designing the most suitable target sequences
for generating Cas9 cleavage-mediated, genetically modified
mice, it is important to avoid targeting sites that have only one
or two mismatches in other genomic loci. This is particularly
important for mismatches that are at the PAM distal region.
We consider several possibilities to explain the lower off-target
cleavage rate seen in animals derived from manipulated zygotes
and the results reported for CRISPR/Cas-treated human cell
lines (Fu et al., 2013; Hsu et al., 2013). (1) In our study, the off-
target mutagenesis was based on the analysis of a ‘clonal
genome’ in animals derived from a single manipulated zygote
in contrast to the previous reports that analyzed heterogeneous
cell populations. The surveyor assay, based upon extensive PCR
amplification, may identify any mutation, even very rare alleles
that may be present in the heterogeneous population. (2) The
transformed human cell lines may have different DNA damage
responses resulting in a different mutagenesis rate than the
normal one-cell embryo. (3) In our experiments CRISPR/Cas
was injecting as short-lived RNA in contrast to Fu et al. (2013)
and Hsu et al. (2013), who used DNA plasmid transfection, which
may express the Cas9/sgRNA for longer time periods leading to
more extensive cleavage. Thus, our data suggest high specificity
of the CRISPR/Cas9 system for gene editing in early embryos
aimed at generating gene-modified mice. Nevertheless, future
characterization of off-target mutagenesis of CRISPR/Cas sys-
tem using whole-genome sequencing would be highly informa-
tive and may allow designing sgRNAs with higher specificity.
In summary, CRISPR/Cas-mediated genome editing repre-
sents an efficient and simple method of generating sophisticated
genetic modifications in mice such as conditional alleles and
endogenous reporters in one step. The principles described in
this study could be directly adapted to other mammalian spe-
cies, opening the possibility of sophisticated genome engineer-
ing in many species where ES cells are not available.
EXPERIMENTAL PROCEDURES
Production of Cas9 mRNA and sgRNA
Bicistronic expression vector px330 expressing Cas9 and sgRNA (Cong et al.,
2013) was digested with BbsI and treated with Antarctic Phosphatase, and the
linearized vector was gel purified. A pair of oligos (Table S4) for each targeting
site was annealed, phosphorylated, and ligated to the linearized vector.
T7 promoter was added to Cas9 coding region by PCR amplification using
primer Cas9 F and R (Table S4). T7-Cas9 PCR product was gel purified and
used as the template for in vitro transcription (IVT) using mMESSAGE
mMACHINE T7 ULTRA kit (Life Technologies). T7 promoter was added to
sgRNAs template by PCR amplification using primer listed in Table S4. The
T7-sgRNA PCR product was gel purified and used as the template for IVT
using MEGAshortscript T7 kit (Life Technologies). Both the Cas9 mRNA and
the sgRNAs were purified using MEGAclear kit (Life Technologies) and eluted
in RNase-free water.
Single-Stranded and Double-Stranded DNA Donors
All single-stranded oligos were ordered as Ultramer DNA oligos from Inte-
grated DNA Technologies. Nanog-2A-mCherry vector was modified from
previously published targeting vector Nanog-2A-mCherry-PGK-Neo (Faddah
8 Cell 154, 1–10, September 12, 2013 ª2013 Elsevier Inc.
Please cite this article in press as: Yang et al., One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Medi-
ated Genome Engineering, Cell (2013), http://dx.doi.org/10.1016/j.cell.2013.08.022
et al., 2013). Nanog-2A-mCherry-PGK-Neo was digested with PacI and AscI to
drop out the PGK-Neo cassette; the 9.7 kb fragment was gel purified and
blunt-ended using T4 DNA polymerase (New England Biolabs) then self-
ligated using T4 DNA ligase (New England Biolabs). Oct4-IRES-eGFP-PGK-
Neo vector is previously published (Lengner et al., 2007).
Suveryor Assay and RFLP Analysis for Genome Modification
Suveryor assay was performed as described (Guschin et al., 2010). Genomic
DNA from targeted and control mice or embryos was extracted and PCR
was performed using gene-specific primers (Table S4) under the following
conditions: 95
!
C for 5 min; 35 3 (95
!
C for 30 s, 60
!
C for 30 s, 68
!
C for 40 s);
68
!
C for 2 min; hold at 4
!
C. PCR products were then denatured, annealed,
and treated with Suveryor nuclease (Transgenomic). DNA concentration of
each band was measured on an ethidium-bromide-stained 10% acrylamide
Criterion TBE gel (BioRad) and quantified using ImageJ software. For RFLP
analysis, 10 ml of Tet1, Tet2, Mecp2-R1, R2 PCR product was digested with
EcoRI, 10 ml of Mecp2-L1, and L2 PCR product was digested with NheI.
Digested DNA was separated on an ethidium-bromide-stained agarose gel
(2%). For sequencing, PCR products were cloned using the Original TA
Cloning Kit (Invitrogen), and mutations were id entified by Sanger sequencing.
One-Cell Embryo Injection
All animal procedures were performed according to NIH guidelines and
approved by the Com mittee on Animal Care at MIT. B6D2F1 (C57BL/6 X
DBA2) female mice and ICR mouse strains were used as embryo donors
and foster mothers, respectively. Superovulated female B6D2F1 mice (7–
8 weeks old) were mated to B6D2F1 stud males, and fertilized embryos
were collected from oviducts. Different concentrations of Cas mRNA, sgRNA,
and oligos or plasmid vectors were mixed and injected into the cytoplasm or
pronucleus of fertilized eggs with well-recognized pronuclei in M2 medium
(Sigma). The injected zygotes were cultured in KSOM with amino acids at
37
!
C under 5% CO2 in air until blastocyst stage by 3.5 days. Thereafter, 15–
25 blastocysts were transferred into uterus of pseudopregnant ICR females
at 2.5 dpc.
Southern Blotting
Genomic DNA was separated on a 0.8% agarose gel after restriction digests
with the appropriate enzymes, transferred to a nylon membrane (Amersham)
and hybridized with
32
P random primer (Stratagene)-labeled probes. Between
hybridizations, blots were stripped and checked for complete removal of
radioactivity before rehybridization with a different probe.
In Vitro Cre Recombination
A 20 ml reaction containing 1 mg of genomic DNA and 10 units of recombinant
Cre recombinase (New England Biolabs) in 13 buffer was incubated at 37
!
C
for 1 hr. For all targets, 1 ml of the Cre reaction mix was used as template for
PCR reactions with gene-specific primers. For each target, primers DF and
DR were used for detecting the deletion products, and primers CF and CR
were used to detect the circle product. All products were sequenced.
Immunostaining and Western Blot Analysis
For immunostaining, cells in 24-well were fixed in PBS supplemented with 4%
paraformaldehyde for 15 min at room temperature (RT). The cells were then
permeabilized using 0.2% Triton X-100 in PBS for 15 min at RT. The cells
were blocked for 30 min in 1% BSA in PBS. Primary antibody against V5
(ab9137, abcam) was diluted in the same blocking buffer and incubated with
the samples overnight at 4
!
C. The cells were treated with a fluorescently
coupled secondary antibody and then incubated for 1 hr at RT. The nuclei
were stained with Hoechst 33342 (Sigma) for 5 min at RT.
For western blot, Cell pellets were lysed on ice in Laemmli buffer (62.5 mM
Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate, 5% beta-mercaptoethanol,
10% glycerol, and 0.01% bromophenol blue) for 30 min in presence of prote-
ase inhibitors (Roche Diagnostics), boiled for 5–7 min at 100
!
C, and subjected
to western blot analysis. Primary antibodies: V5 (1:1,000, ab9137, Abcam),
beta-actin (1:2,000). Blots were probed with anti-goat, or anti-rabbit IgG-
HRP secondary antibody (1:10,000) and visualized using ECL detection kit
(GE Healthcare).
ESC Derivation and Differentiation
Morulas or blastocysts were selected to generate ES cell lines. The zona
pellucida was removed using acid Tyrode solution. Each embryo was trans-
ferred into one well of a 96-well plate seeded with ICR embryonic fibroblast
feeders in ESC medium supplemented with 20% knockout serum replace-
ment, 1,500 U/ml leukemia inhibitory factor (LIF), and 50 mM of the MEK1 inhib-
itor (PD098059). After 4–5 days in culture, the colonies were trypsinized and
transferred to a 96-well plate with a fresh feeder layer in fresh medium. Clonal
expansion of the ESCs proceeded from 48-well plates to 6-well plates with
feeder cells and then to 6-well plates for routine culture.
For ESC differentiation, cells were harvested by trypsinization and trans-
ferred to bacterial culture dishes in the ES medium without or LIF. After
3 days, aggregated cells were plated onto gelatin-coated tissue culture dishes
and incubated for another 3 days.
Prediction of Potential Off-Targets
Potential off-targets were predicted by searching the mouse genome (mm9)
for matches to the 20 nt sgRNA sequence allowing for up to four mismatches
(Nanog) or three mismatches (Sox2, Oct4, Mecp2-L2, and Mecp2-R1) fol-
lowed by NGG PAM sequence. Matches were ranked first by ascending
number of mismatches then by ascending distance from the PAM sequence.
SUPPLEMENTAL INFORMATION
Supplemental Information includes four figures, and four tables and can be
found with this article online at http://dx.doi.org/10.1016/j.cell.2013.08.022.
ACKNOWLEDGMENTS
We thank Ruth Flannery and Kibibi Ganz for support with animal care and ex-
periments. We thank Jaenisch lab members for helpful discussions on the
manuscript. We are also grateful to Dina A. Faddah for help with editing the
manuscript. A.W.C. is supported by a Croucher scholarship. R.J. is an adviser
to Stemgent and a cofounder of Fate Therapeutics. This work was supported
by NIH grants HD 045022 and R37CA084198 to R.J.
Received: July 25, 2013
Revised: August 12, 2013
Accepted: August 14, 2013
Published: August 29, 2013
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ated Genome Engineering, Cell (2013), http://dx.doi.org/10.1016/j.cell.2013.08.022
    • "Our strategy for correcting this allele involved directly injecting CRISPR reagents into one-cell stage mouse embryos. This approach has previously been shown to introduce subtle modifications into the genome at high efficiency [20, 21]. Given reported concerns regarding the specificity of the Cas9 nuclease and potential off-target effects, we opted to use paired offset guides along with a nickase version (D10A) of the Cas9 protein to correct the B6N.Cdh23 ahl allele [22, 23]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Nuclease-based technologies have been developed that enable targeting of specific DNA sequences directly in the zygote. These approaches provide an opportunity to modify the genomes of inbred mice, and allow the removal of strain-specific mutations that confound phenotypic assessment. One such mutation is the Cdh23 (ahl) allele, present in several commonly used inbred mouse strains, which predisposes to age-related progressive hearing loss. Results: We have used targeted CRISPR/Cas9-mediated homology directed repair (HDR) to correct the Cdh23 (ahl) allele directly in C57BL/6NTac zygotes. Employing offset-nicking Cas9 (D10A) nickase with paired RNA guides and a single-stranded oligonucleotide donor template we show that allele repair was successfully achieved. To investigate potential Cas9-mediated 'off-target' mutations in our corrected mouse, we undertook whole-genome sequencing and assessed the 'off-target' sites predicted for the guide RNAs (≤4 nucleotide mis-matches). No induced sequence changes were identified at any of these sites. Correction of the progressive hearing loss phenotype was demonstrated using auditory-evoked brainstem response testing of mice at 24 and 36 weeks of age, and rescue of the progressive loss of sensory hair cell stereocilia bundles was confirmed using scanning electron microscopy of dissected cochleae from 36-week-old mice. Conclusions: CRISPR/Cas9-mediated HDR has been successfully utilised to efficiently correct the Cdh23 (ahl) allele in C57BL/6NTac mice, and rescue the associated auditory phenotype. The corrected mice described in this report will allow age-related auditory phenotyping studies to be undertaken using C57BL/6NTac-derived models, such as those generated by the International Mouse Phenotyping Consortium (IMPC) programme.
    Full-text · Article · Dec 2016
    • "To generate the Oct4:IRES:EGFP reporter line used in Additional file 1: Figure S1A, a published guide RNA (gRNA) and plasmid were used to target CJ9 ESCs [27] and insert an IRES:EGFP cassette into the 3′ UTR of Oct4. Briefly, a guide RNA specific to the 3′ UTR of the Oct4 locus (CTCAGTGATGCTGTTGATC) was cloned into the Cas9-expressing vector px459 [69]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: The cohesin complex consists of multiple core subunits that play critical roles in mitosis and transcriptional regulation. The cohesin-associated protein Wapal plays a central role in off-loading cohesin to facilitate sister chromatid separation, but its role in regulating mammalian gene expression is not understood. We used embryonic stem cells as a model, given that the well-defined transcriptional regulatory circuits were established through master transcription factors and epigenetic pathways that regulate their ability to maintain a pluripotent state. Results: RNAi-mediated depletion of Wapal causes a loss of pluripotency, phenocopying loss of core cohesin subunits. Using chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq), we determine that Wapal occupies genomic sites distal to genes in combination with CTCF and core cohesin subunits such as Rad21. Interestingly, genomic sites occupied by Wapal appear enriched for cohesin, implying that Wapal does not off-load cohesin at regions it occupies. Wapal depletion induces derepression of Polycomb group (PcG) target genes without altering total levels of Polycomb-mediated histone modifications, implying that PcG enzymatic activity is preserved. By integrating ChIP-seq and gene expression changes data, we identify that Wapal binding is enriched at the promoters of PcG-silenced genes and is required for proper Polycomb repressive complex 2 (PRC2) recruitment. Lastly, we demonstrate that Wapal is required for the interaction of a distal cis-regulatory element (CRE) with the c-Fos promoter. Conclusions: Collectively, this work indicates that Wapal plays a critical role in silencing of PcG target genes through the interaction of distal CREs with promoters.
    Full-text · Article · Dec 2016
    • "In mammalian cells, total homology up to 10 kb increases the HDR frequencies without even a target cleavage, an observation that has been widely used for creating gene-targeting vectors for the generation of animal models (Deng and Capecchi 1992). In the presence of an induced DSB at the target site, a plasmid donor with at least 1-2 kb of total homology is recommended for large sequence changes more than 100 bp (Dickinson, Ward et al. 2013, Yang, Wang et al. 2013). The cleavage site should be as close to the insertion site of the mutation as possible, ideally less as 10 bp away. "
    [Show abstract] [Hide abstract] ABSTRACT: In vivo genome editing represents an emerging field in the treatment of monogenic disorders, as it may constitute a solution to the current hurdles in classic gene addition therapy, which are the low levels and limited duration of transgene expression. Following the introduction of a double strand break (DSB) at the mutational site by highly specific endonucleases, such as TALENs (transcription activator like effector nucleases) or RNA based nucleases (clustered regulatory interspaced short palindromic repeats - CRISPR-Cas), the cell's own DNA repair machinery restores integrity to the DNA strand and corrects the mutant sequence, thus allowing the cell to produce protein levels as needed. The DNA repair happens either through the error prone non-homologous end-joining (NHEJ) pathway or with high fidelity through homology directed repair (HDR) in the presence of a DNA donor template. A third pathway called microhomology mediated endjoining (MMEJ) has been recently discovered. In this review, the authors focus on the different DNA repair mechanisms, the current state of the art tools for genome editing and the particularities of the retina and photoreceptors with regard to in vivo therapeutic approaches. Finally, current attempts in the field of retinal in vivo genome editing are discussed and future directions of research identified.
    Full-text · Article · Sep 2016
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