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Editorial
Genome Editing of a CArG Element in the Mouse
Genome Establishes its Role in Gene Expression
Kiran Musunuru
I
n the February issue, Han et al1 present the use of a novel
clustered regularly interspaced short palindromic repeat
(CRISPR)-CRISPR-associated 9 (Cas9) method to inactivate the calponin-1 gene involved in smooth muscle contraction, by introducing a knockin mutation to cleanly disrupt a
CC(A/T)6GG (CArG) element in the first intron of the gene.
Previous work by the group had suggested a critical role of
the CArG element in calponin-1 gene expression in both
humans and mice.2 Here, the authors injected the components
of CRISPR-Cas9—an mRNA for the Cas9 endonuclease, a
guide RNA containing the 20-nucleotide sequence matching the sequence of the CArG element, and a 135-nucleotide
single-strand DNA oligonucleotide overlapping the CArG
element and containing the desired knockin mutation—into
the cytoplasm of fertilized mouse eggs (Figure). This resulted
in stable incorporation of the CArG knockin mutation into
the genome in some of the eggs. After implantation of the
resulting blastocysts into surrogate mothers, the injected
eggs yielded founder mice in 3 weeks. Remarkably, 3 of 18
founder mice (17%) carried the desired knockin mutation
on ≥1 chromosome, with 1 founder having the mutation on
both chromosomes. The founder mice were successfully bred,
with germline transmission of the knockin mutation. With
calponin-1 being a smooth muscle cell–restricted gene, the
authors examined smooth muscle tissues from wild-type, heterozygous, and homozygous mice and found almost complete
suppression of calponin-1 gene expression in the homozygous
mice. This simple yet elegant experiment, performed within
a time frame of a few months, unequivocally establishes the
critical role of the CArG element in calponin-1 gene expression in smooth muscle tissues.
See accompanying article on page 312
in the February 2015 issue
To fully appreciate the game-changing nature of CRISPRCas9, consider a similar study performed in the pregenomeediting era. Khromov et al3 engineered a knockin mouse
in which a 30-nucleotide fragment containing an AT-rich
sequence and a CArG element was deleted to suppress telokin
gene expression in smooth muscle tissues. This entailed a long
series of steps. First, a double-strand targeting vector containing 6-kb and 3-kb homology arms flanking an antibiotic
resistance cassette (replacing the 30-nucleotide fragment and
flanked by loxP sequences) was produced. Second, the targeting vector was electroporated into mouse embryonic stem
cells, which were selected with antibiotic and then expanded.
Third, mouse embryonic stem cell clones were screened for
correct insertion of the antibiotic resistance cassette into the
telokin locus. Fourth, embryonic stem cells were injected into
blastocysts and implanted into surrogate mothers to yield chimeric mice. Fifth, the chimeric mice were bred to obtain mice
that had inherited the mutant allele through the germline. Sixth,
as part of the breeding, male mice expressing Cre recombinase
in the germline were used to remove the antibiotic resistance
cassette. Finally, the floxed alleles were bred to homozygosity
to yield the final mice for study. A conservative estimate of the
amount of time required to carry out all these steps is ≥1 year
and probably ≈2 years; yet, despite all this effort, the end result
was a mutant allele in which the 30-nucleotide CArG-bearing
fragment was replaced with a 34-nucleotide loxP sequence,
effective but crude. In contrast, Han et al1 were able to carry
out their study in just a few months while creating a more subtle mutant allele in which several nucleotides were substituted
to impair the CArG element, with no need for antibiotic resistance, the Cre-loxP system, and so on.
The tremendous use of CRISPR-Cas9 in generating knockout and knockin mice was initially demonstrated by Rudolf
Jaenisch’s group4,5 and has since been validated with its ability to
correct pathogenic mutations in mouse embryos6,7 and to generate knockout and knockin mutations in a wide variety of model
organisms, most impressively in monkeys.8 The work by Han
et al1 represents the first case in which CRISPR-Cas9 has been
used to manipulate smooth muscle gene expression by editing a
key noncoding regulatory element and can easily be extended to
rapidly and efficiently interrogate the function of any number of
noncoding regulatory elements throughout the mouse genome.
This successful demonstration of the method should have a
long-time impact not only in the field of smooth muscle biology
but also in many other fields of biomedical research.
Disclosures
From the Department of Stem Cell and Regenerative Biology, Harvard
University, Cambridge, MA; and Division of Cardiovascular Medicine,
Brigham and Women’s Hospital, Boston, MA.
Correspondence to Kiran Musunuru, MD, PhD, MPH, Harvard
University, 7 Divinity Ave, Cambridge, MA 02138. E-mail
[email protected]
(Arterioscler Thromb Vasc Biol. 2015;35:496-497.
DOI: 10.1161/ATVBAHA.115.305175.)
© 2015 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
DOI: 10.1161/ATVBAHA.115.305175
None.
References
1. Han Y, Slivano OJ, Christie CK, Cheng AW, Miano JM. CRISPR-Cas9
genome editing of a single regulatory element nearly abolishes target
gene expression in mice––brief report. Arterioscler Thromb Vasc Biol.
2015;35:312–315. doi: 10.1161/ATVBAHA.114.305017.
2.Long X, Slivano OJ, Cowan SL, Georger MA, Lee TH, Miano JM.
Smooth muscle calponin: an unconventional CArG-dependent gene
that antagonizes neointimal formation. Arterioscler Thromb Vasc Biol.
2011;31:2172–2180. doi: 10.1161/ATVBAHA.111.232785.
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at UNIV OF ROCHESTER on April 14, 2015
496
Musunuru CArG Element in the Mouse Genome 497 AQ3
Figure. Accelerated generation of knockin mice with clustered
regularly interspaced short palindromic repeat (CRISPR)CRISPR-associated 9 (Cas9). PCR indicates polymerase chain
reaction.
3. Khromov AS, Wang H, Choudhury N, McDuffie M, Herring BP, Nakamoto
R, Owens GK, Somlyo AP, Somlyo AV. Smooth muscle of telokin-deficient
mice exhibits increased sensitivity to Ca2+ and decreased cGMP-induced
relaxation. Proc Natl Acad Sci U S A. 2006;103:2440–2445. doi: 10.1073/
pnas.0508566103.
4. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch
R. One-step generation of mice carrying mutations in multiple genes by
CRISPR/Cas-mediated genome engineering. Cell. 2013;153:910–918.
doi: 10.1016/j.cell.2013.04.025.
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Cas-mediated genome engineering. Cell. 2013;154:1370–1379. doi:
10.1016/j.cell.2013.08.022.
6. Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, Yan Z, Li D, Li J.
Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell
Stem Cell. 2013;13:659–662. doi: 10.1016/j.stem.2013.10.016.
7. Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson
EN. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated
editing of germline DNA. Science. 2014;345:1184–1188. doi: 10.1126/
science.1254445.
8. Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cynomolgus
monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell.
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Key Words: gene expression
transgenic mice
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gene knockout
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smooth muscle
Genome Editing of a CArG Element in the Mouse Genome Establishes its Role in Gene
Expression
Kiran Musunuru
Arterioscler Thromb Vasc Biol. 2015;35:496-497
doi: 10.1161/ATVBAHA.115.305175
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