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Fighting invasion. When
viruses (green) attack
bacteria, the bacteria
respond with DNA-targeting
defenses that biologists
have learned to exploit
for genetic engineering.
The CRISPR Craze
CREDIT: EYE OF SCIENCE/SCIENCE SOURCE
A bacterial immune system yields a potentially
revolutionary genome-editing technique
BACTERIA MAY NOT ELICIT MUCH SYMPAthy from us eukaryotes, but they, too, can get
sick. That’s potentially a big problem for the
dairy industry, which often depends on bacteria such as Streptococcus thermophilus to
make yogurts and cheeses. S. thermophilus
breaks down the milk sugar lactose into tangy
lactic acid. But certain viruses—bacteriophages, or simply phages—can debilitate the
bacterium, wreaking havoc on the quality or
quantity of the food it helps produce.
In 2007, scientists from Danisco, a
Copenhagen-based food ingredient com-
pany now owned by DuPont, found a way to
boost the phage defenses of this workhouse
microbe. They exposed the bacterium to
a phage and showed that this essentially
vaccinated it against that virus (Science,
23 March 2007, p. 1650). The trick has
enabled DuPont to create heartier bacterial
strains for food production. It also revealed
something fundamental: Bacteria have a
kind of adaptive immune system, which
enables them to fight off repeated attacks
by specific phages.
That immune system has suddenly
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become important for more than food scientists and microbiologists, because of a valuable feature: It takes aim at specific DNA
sequences. In January, four research teams
reported harnessing the system, called
CRISPR for peculiar features in the DNA of
bacteria that deploy it, to target the destruction of specific genes in human cells. And in
the following 8 months, various groups have
used it to delete, add, activate, or suppress targeted genes in human cells, mice, rats, zebrafish, bacteria, fruit flies, yeast, nematodes,
and crops, demonstrating broad utility for the
23 AUGUST 2013
833
technique. Biologists had recently developed
several new ways to precisely manipulate
genes, but CRISPR’s “efficiency and ease of
use trumps just about anything,” says George
Church of Harvard University, whose lab was
among the first to show that the technique
worked in human cells.
With CRISPR, scientists can create
mouse models of human diseases much
more quickly than before, study individual
genes much faster, and easily change multiple genes in cells at once to study their
interactions. This year’s CRISPR craze may
yet slow down as limitations of the method
emerge, but Church and other CRISPR pioneers are already forming companies to
harness the technology for treating genetic
diseases. “I don’t think there’s any example
of any field moving this fast,” says Blake
Wiedenheft, a biochemist at Montana State
University in Bozeman.
Humble beginnings
The first inkling of this hot new genetic engineering tool came in 1987, when a research
team observed an oddly repetitive sequence at
one end of a bacterial gene. Few others took
much notice. A decade later, though, biologists
deciphering microbial genomes often found
similar puzzling patterns, in which a sequence
of DNA would be followed by nearly the same
sequence in reverse, then 30 or so seemingly
random bases of “spacer DNA,” and then a
repeat of the same palindromic sequence, followed by a different spacer DNA. A single
microbe could have several such stretches,
each with different repeat and intervening
sequences. This pattern appears in more than
40% of bacteria and fully 90% of microbes
in a different domain, the archaea, and gives
CRISPR its name. (It stands for clustered regularly interspaced short palindromic repeats.)
Many researchers assumed that these
odd sequences were junk, but in 2005, three
bioinformatics groups reported that spacer
DNA often matched the sequences of phages,
indicating a possible role for CRISPR in
microbial immunity. “That was a very key
clue,” says biochemist Jennifer Doudna of the
University of California (UC), Berkeley. It
led Eugene Koonin from the National Center
for Biotechnology Information in Bethesda,
Maryland, and his colleagues to propose that
bacteria and archaea take up phage DNA,
then preserve it as a template for molecules
of RNA that can stop matching foreign DNA
in its tracks, much the way eukaryotic cells
use a system called RNA interference (RNAi)
to destroy RNA.
Enter the Danisco team. In 2007,
Rodolphe Barrangou, Philippe Horvath, and
834
Precise cuts. In just 8 months, CRISPR modifications of DNA resulted in dumpier nematodes (top,
bottom), zebrafish embryos with an excess of ventral
tissue (middle, bottom), and fruit flies with dark eyes
(bottom, right), demonstrating its broad utility for
editing genes in animals.
others with the company showed that they
could alter the resistance of S. thermophilus
to phage attack by adding or deleting
spacer DNA that matched the phage’s. At
the time, Barrangou, who is now at North
Carolina State University in Raleigh, didn’t
see CRISPR’s full potential. “We had no idea
that those elements could be readily exploitable for something as attractive as genome
editing,” he says.
Doudna and Emmanuelle Charpentier,
currently of the Helmholtz Centre for Infection Research and Hannover Medical School
23 AUGUST 2013
in Germany, took the next step. They had
independently been teasing out the roles of
various CRISPR-associated proteins to learn
how bacteria deploy the DNA spacers in their
immune defenses. But the duo soon joined
forces to focus on a CRISPR system that
relies on a protein called Cas9, as it was simpler than other CRISPR systems.
When CRISPR goes into action in
response to an invading phage, bacteria
transcribe the spacers and the palindromic
DNA into a long RNA molecule that the cell
then cuts into short spacer-derived RNAs
called crRNAs. An additional stretch of
RNA, called tracrRNA, works with Cas9
to produce the crRNA, Charpentier’s group
reported in Nature in 2011. The group proposed that together, Cas9, tracrRNA, and
crRNA somehow attack foreign DNA that
matches the crRNA.
The two teams found that the Cas9 protein is a nuclease, an enzyme specialized for
cutting DNA, with two active cutting sites,
one site for each strand of the DNA’s double
helix. And in a discovery that foreshadowed
CRISPR’s broad potential for genome engineering, the team demonstrated that they
could disable one or both cutting sites without interfering with the ability of the complex to home in on its target DNA. “The
possibility of using a single enzyme by just
changing the RNA seemed very simple,”
Doudna recalls.
Before CRISPR could be put to use,
however, Doudna’s and Charpentier’s teams
had to show that they could control where
Cas9 went to do its cutting. First, Doudna’s
postdoc, Martin Jinek, figured out how to
combine tracrRNA and spacer RNA into a
“single-guide RNA” molecule; then, as a
proof of principle, the team last year made
several guide RNAs, mixed them with Cas9,
and showed in a test tube that the synthetic
complexes could find and cut their DNA targets (Science, 17 August 2012, p. 816). “That
was a milestone paper,” Barrangou says.
This precision targeting drives the
growing interest in CRISPR. Genetic engineers have long been able to add and delete
genes in a number of organisms. But they
couldn’t dictate where those genes would
insert into the genome or control where gene
deletions occurred. Then, a decade ago,
researchers developed zinc finger nucleases,
synthetic proteins that have DNA-binding
domains that enable them to home in and
break DNA at specific spots. A welcome
addition to the genetic engineering toolbox,
zinc fingers even spawned a company that is
testing a zinc finger to treat people infected
with HIV (Science, 23 December 2005,
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CREDITS (TOP TO BOTTOM): FRIEDLAND ET AL., NATURE METHODS 10 (JUNE 2013); ANDREW GONZALES/JOANNA YEH; SCOTT GRATZ/UNIVERSITY OF WISCONSIN, MADISON
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p. 1894). More recently, synthetic nucleases
Such work lays the foundation for
called TALENs have proved an easier way to generating mutant mice, a key tool for biotarget specific DNA and were predicted to medical research. One approach would be to
surpass zinc fingers (Science, 14 December add the altered mouse ES cells to a develop2012, p. 1408).
ing embryo and breed the resulting animals.
Now, CRISPR systems have stormed But Zhang has demonstrated a faster option.
onto the scene, promising to even out- His team found it could simply inject fertilcompete TALENs. Unlike the CRISPR sys- ized mouse eggs, or zygotes, with Cas9 mestem, which uses RNA as its DNA-homing senger RNA and two guide RNAs and, with
mechanism, zinc finger and TALEN tech- 80% efficiency, knock out two genes. They
nologies both depend on custom-making new could also perform more delicate genomic
proteins for each DNA target.
The CRISPR system’s “guide
Cas9
RNAs” are much easier to make
Guide RNA
than proteins, Barrangou says.
“Within a couple weeks you can
Active
sites
generate very tangible results
*
that using alternative methods
would take months.”
Target specific
crRNA sequence
weeks. And Zhang thinks the approach is not
limited to mice. “As long as you can manipulate the embryo and then reimplant it, then
you will be able to do it” in larger animals,
perhaps even primates.
Doudna’s group and a Korean team
reported using CRISPR to cut DNA in
human cells 3 weeks after Zhang’s and
Church’s papers went online, and, at the same
time, another group revealed they had used
CRISPR to make mutant zebrafish. This cas-
Cas9
*
*
Harnessing CRISPR
*
Speed is not its only advantage. Church’s group had been
pushing the use of TALENs in
Target DNA
sequence
human cells, but when he learned
of Doudna and Charpentier’s
results, he and his colleagues
made guide RNA against genes
they had already targeted with
TALENs. In three human cell
types, the CRISPR system was
*
more efficient than TALENs
at cutting the DNA target, and
*
it worked on more genes than
TALENs did (Science, 15 February, p. 823). To demonstrate
Activator
Repressor
the ease of the CRISPR system, Church’s team synthesized
Deactivated
Deactivated
a library of tens of thousands
Cas9
Cas9
of guide RNA sequences, capable of targeting 90% of human
genes. “You can pepper the
genome with every imaginable
Target gene
mRNA
CRISPR,” he says.
That makes it possible to
alter virtually any gene with
Cas9, exploiting its DNA- DNA surgeon. With just a guide RNA and a protein called Cas9, researchers first showed that the CRISPR system can home
cutting ability to either disable the in on and cut specific DNA, knocking out a gene or enabling part of it to be replaced by substitute DNA. More recently, Cas9
gene or cut it apart, allowing sub- modifications have made possible the repression (lower left) or activation (lower right) of specific genes.
stitute DNA to be inserted. In an
independent paper that appeared at the same surgery on the embryos by shackling Cas9, cade of papers has had a synergistic effect,
time as Church’s, Feng Zhang, a synthetic so that it nicks target DNA instead of cutting commanding the attention of a broad swath
biologist at the Broad Institute in Cambridge, it. In this way, they could introduce a new part of the biology community. “If a single paper
Massachusetts, and his colleagues showed of a gene through a process called homology- comes out, it gets some attention, but when
that CRISPR can target and cut two genes at directed repair, they reported in the 2 May six papers come out all together, that’s when
once in human cells (Science, 15 February, issue of Cell.
people say, ‘I have to do this,’ ” says Charles
p. 819). And working with developmental
Developing a new mouse model for a Gersbach, a biomedical engineer at Duke
biologist Rudolf Jaenisch at the Whitehead disease now entails careful breeding of mul- University in Durham, North Carolina.
Institute for Biomedical Research in Cam- tiple generations and can take a year; with
Once she saw Doudna and Charpentier’s
bridge, Zhang has since disrupted five genes Zhang’s CRISPR technique, a new mouse paper a year ago, Gao Caixia became one of
at once in mouse embryonic stem (ES) cells.
model could be ready for testing in a matter of the early converts. Her group at the Chinese
CREDIT: K. SUTLIFF/SCIENCE
CRISPR in Action
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Academy of Sciences’ Institute of Genetics
and Developmental Biology in Beijing had
been using zinc finger and TALENs technology on rice and wheat. Using CRISPR, they
have now disabled four rice genes, suggesting
that the technique could be used to engineer
this crucial food crop. In wheat, they knocked
bacteria, the presence of Cas9 alone is enough
to block transcription, but for mammalian
applications, Qi and colleagues add to it a section of protein that represses gene activity. Its
guide RNA is designed to home in on regulatory DNA, called promoters, which immediately precede the gene target.
CRISPR technology may yet have limitations. It’s unclear, for example, how specific the guide RNAs are for just the genes
they are supposed to target. “Our initial data
suggest that there can be significant offtarget effects,” says J. Keith Joung from the
Massachusetts General Hospital in Boston, who back in January demonstrated
that CRISPR would alter genes in zebrafish
embryos and has used CRISPR to turn on
genes. His work shows that nontarget DNA
resembling the guide RNA can become cut,
activated, or deactivated.
Joung’s group showed that a guide RNA
can target DNA that differs from the intended
target sequence in up to five of its bases.
Zhang has gotten more reassuring results but
says that “the specificity is still something we
have to work on,” especially as more people
begin to think about delivering CRISPR systems as treatments for human diseases. “To
really make the technology safe, we really
have to make sure it goes where we want it to
go to and nowhere else.”
Researchers must also get the CRISPR
CRISPRed rice. Earlier this month, researchers showed CRISPR works in plants, such as rice, where the
knocked-out gene resulted in dwarf albino individuals (right).
components to the right place. “Delivery
is an enormous challenge and will be cell
out a gene that, when disabled, may lead to
Last month, that team and three other type and organism specific,” Joung notes.
plants resistant to powdery mildew. In a mea- groups used a Cas9 to ferry a synthetic With zebrafish, his team injects guide
sure of the excitement that CRISPR has gen- transcription factor—a protein fragment RNA and messenger RNA for Cas9 directly
erated, the team’s report in the August issue that turns on genes—enabling them to acti- into embryos; with mammalian cells,
of Nature Biotechnology was accompanied vate specific human genes. Just using one they use DNA constructs. How CRISPR
by four other papers describing CRISPR suc- CRISPR construct had a weak effect, but might be delivered into adult animals,
cesses in plants and in rats.
all four teams found a way to amplify it. or to treat disease in people, is just now
The cost of admission is low: Free soft- By targeting multiple CRISPR constructs being considered.
ware exists to design guide RNA to target to slightly different spots on the gene’s proUltimately, CRISPR may take a place
any desired gene, and a repository called moter, says Gersbach, one of the team lead- beside zinc fingers and TALENs, with the
Addgene, based in Cambridge, offers aca- ers, “we saw a huge synergistic effect.”
choice of editing tool depending on the
demics the DNA to make their own
particular application. But for now,
CRISPR system for $65. Since the
researchers are dazzled by the ease by
beginning of the year, Addgene—
which they can make and test different
to which 11 teams have contributed
CRISPR variants and by the technoloCRISPR-enabling DNA sequences—
gy’s unexplored potential. Charpentier
has distributed 5000 CRISPR conand others are looking at the versions
structs, and in a single July week
of Cas9 in other bacteria that might
the repository received 100 orders
work better than the one now being
for a new construct. “They are kind
used. Microbiologists have harnessed
—Blake Wiedenheft,
of crazy hot,” says Joanne Kamens,
the CRISPR system to vaccinate bacMontana State University
Addgene’s executive director.
teria against the spread of antibiotic
resistance genes. Church, Doudna,
Fine-tuning gene activity
Charpentier, and others are forming
The initial CRISPR genome-editing papers all
In the 25 July issue of Nature Meth- CRISPR-related companies to begin explorrelied on DNA cutting, but other applications ods, he reported activating genes tied to ing human therapeutic applications, includquickly appeared. Working with Doudna, Lei human diseases, including those involved in ing gene therapy.
S. Qi from UC San Francisco and his col- muscle differentiation, controlling canAnd there’s more that can be done,
leagues introduced “CRISPRi,” which, like cer and inflammation, and producing fetal Barrangou says. “The only limitation today
RNAi, turns off genes in a reversible fashion hemoglobin. Two other teams also targeted is people’s ability to think of creative ways to
and should be useful for studies of gene func- biomedically important genes. CRISPR harness [CRISPR].”
tion. They modified Cas9 so it and the asso- control of such genes could treat diseases
Not bad for a system that started with
ciated guide RNA would still home in on a ranging from sickle cell anemia to arthritis, sickly bacteria.
target but would not cut DNA once there. In Gersbach suggests.
–ELIZABETH PENNISI
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CREDIT: GAO CAIXIA LABORATORY
“I don’t think there’s any
example of any field
moving this fast.”