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A CRISPR method for genome editing
01-22-2013
EDIT CONNECT
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CAMBRIDGE, Mass.—A
research group composed of scientists
from the Massachusetts Institute of
Technology (MIT), the Broad Institute and
Rockefeller University have developed a new technique for precisely
altering
the genomes of living cells by adding or deleting genes, a method that eases
some of the challenges that currently make genome editing
difficult.
Although genome-editing technologies such as designer zinc
fingers, transcription
activator-like effectors and homing meganucleases have
begun to enable targeted genome modifications, there remains a need for new
technologies that
are scalable, affordable and easy to engineer. One such
method, known as homologous recombination, involves delivering a piece of DNA
that includes the
gene of interest flanked by sequences that match the genome
region where the gene is to be inserted. However, this technique's success rate
is very
low because the natural recombination process is rare in normal cells.
More recently, biologists
discovered that they could improve
the efficiency of this process by adding enzymes called nucleases, which can
cut DNA. Zinc fingers are commonly used
to deliver the nuclease to a specific
location, but zinc finger arrays can't target every possible sequence of DNA,
limiting their usefulness.
Furthermore, assembling the proteins is a
labor-intensive and expensive process.
Complexes known as transcription activator-
like effector
nucleases (TALENs) can also cut the genome in specific locations, but these
complexes can also be expensive and difficult to assemble.
According to the researchers, who described their technique
in a recent Science article,
their
system enables researchers to alter several genome sites simultaneously with
much greater control over where new genes are inserted—and at a
lower price
point—which could yield better designed animal models to study human disease as
well as new therapies.
"My original goal was to be able to modify the genome of
animal and human cells so we can more easily make changes," says Dr. Feng
Zhang, an assistant professor of neuroscience at MIT and leader of the research
team who worked on the development of TALENs in his postdoctorate work
at
Harvard University.
To do that, Zhang and his colleagues co-opted clustered
regularly interspaced short palindromic repeats, or CRISPRs, genome-
editing
technologies first discovered by a group of researchers in Japan in 1987. The
scientists found what they termed an "unusual structure" in the
genome of E. coli, consisting of a series of
repeated stretches, interrupted by unique "spacer" sequences. The role of these
sequences was
at first a mystery, but over the years, scientists have come to
understand that the spacer sequences corresponded with phages that had
previously
infected the bacterial cells.
"We took inspiration from the way the system worked in
bacterial
cells, and set out to find out how to transplant the bacterial system
into a mammalian cell," says Zhang.
Making use of naturally occurring bacterial protein-RNA
systems that recognize and snip viral DNA, the researchers created
DNA-editing
complexes that include a nuclease called Cas9 bound to short RNA sequences.
These sequences are designed to target specific locations in
the genome; when
they encounter a match, Cas9 cuts the DNA.
Zhang and his team engineered two
different type II CRISPR
systems and demonstrated that Cas9 nucleases can be directed by short RNAs to
induce precise cleavage at endogenous genomic
loci in human and mouse cells.
Cas9 can also be converted into a nicking enzyme to facilitate
homology-directed repair with minimal mutagenic activity.
Finally, multiple
guide sequences can be encoded into a single CRISPR array to enable
simultaneous editing of several sites within the mammalian
genome,
demonstrating easy programmability and wide applicability of the CRISPR
technology. Each of the RNA segments can target a different sequence.
"That's the beauty of this—you can easily program a nuclease
to target one or more positions
in the genome," Zhang says.
Although for this study, the researchers tested the system
in cells grown in the lab, they now
plan to apply the new technology to study
brain function and diseases.
The new technique has
broad application potential, says
Zhang. The system could be used to design new therapies for diseases such as
Huntington's disease, cystic fibrosis,
autism, diabetes, neurodegenerative
diseases—any medical condition caused by a genetic mutation. The system might
also be useful for treating HIV by
removing patients' lymphocytes and mutating
the CCR5 receptor, through which the virus enters cells. After being put back
in the patient, such cells
would resist infection. And of course, as Zhang
points out, the approach could also make it easier to study human disease by
inducing specific
mutations in human stem cells.
"Using this genome editing system, you can very
systematically
put in individual mutations and differentiate the stem cells
into neurons or cardiomyocytes and see how the mutations alter the biology of
the cells,"
he says. "Anything that requires engineering of an organism to put
in new genes or to modify what's in the genome will be able to benefit from
this.
"
The study, "Multiplex Genome Engineering Using CRISPR/Cas
Systems," was published Jan. 3 in
Science
Express, an electronic
publication of the American Association for the
Advancement of
Science journal Science.
Lead authors of the paper are graduate students Le Cong and Ann Ran. Funding
came from a variety of sources,
including, notably, newscaster Jane Pauley, as
well as the U.S. National Institute of Mental Health; the W.M. Keck
Foundation;
the McKnight Foundation; the Bill & Melinda Gates Foundation; the Damon
Runyon Cancer Research Foundation; the Searle Scholars Program; and MIT alumni
Mike Boylan and Bob
Metcalfe. Code: E01231303 Back |
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