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A CRISPR method for genome editing
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.