CAMBRIDGE, Mass.—A research group composed of scientistsfrom the Massachusetts Institute of Technology (MIT), the Broad Institute andRockefeller University have developed a new technique for precisely alteringthe genomes of living cells by adding or deleting genes, a method that easessome of the challenges that currently make genome editing difficult.
Although genome-editing technologies such as designer zincfingers, transcription activator-like effectors and homing meganucleases havebegun to enable targeted genome modifications, there remains a need for newtechnologies that are scalable, affordable and easy to engineer. One suchmethod, known as homologous recombination, involves delivering a piece of DNAthat includes the gene of interest flanked by sequences that match the genomeregion where the gene is to be inserted. However, this technique's success rateis very low because the natural recombination process is rare in normal cells.
More recently, biologists discovered that they could improvethe efficiency of this process by adding enzymes called nucleases, which cancut DNA. Zinc fingers are commonly used to deliver the nuclease to a specificlocation, but zinc finger arrays can't target every possible sequence of DNA,limiting their usefulness. Furthermore, assembling the proteins is alabor-intensive and expensive process.
Complexes known as transcription activator-like effectornucleases (TALENs) can also cut the genome in specific locations, but thesecomplexes can also be expensive and difficult to assemble.
According to the researchers, who described their techniquein a recent Science article, theirsystem enables researchers to alter several genome sites simultaneously withmuch greater control over where new genes are inserted—and at a lower pricepoint—which could yield better designed animal models to study human disease aswell as new therapies.
"My original goal was to be able to modify the genome ofanimal and human cells so we can more easily make changes," says Dr. FengZhang, an assistant professor of neuroscience at MIT and leader of the researchteam who worked on the development of TALENs in his postdoctorate work atHarvard University.
To do that, Zhang and his colleagues co-opted clusteredregularly interspaced short palindromic repeats, or CRISPRs, genome-editingtechnologies first discovered by a group of researchers in Japan in 1987. Thescientists found what they termed an "unusual structure" in the genome of E. coli, consisting of a series ofrepeated stretches, interrupted by unique "spacer" sequences. The role of thesesequences was at first a mystery, but over the years, scientists have come tounderstand that the spacer sequences corresponded with phages that hadpreviously infected the bacterial cells.
"We took inspiration from the way the system worked inbacterial cells, and set out to find out how to transplant the bacterial systeminto a mammalian cell," says Zhang.
Making use of naturally occurring bacterial protein-RNAsystems that recognize and snip viral DNA, the researchers created DNA-editingcomplexes that include a nuclease called Cas9 bound to short RNA sequences.These sequences are designed to target specific locations in the genome; whenthey encounter a match, Cas9 cuts the DNA.
Zhang and his team engineered two different type II CRISPRsystems and demonstrated that Cas9 nucleases can be directed by short RNAs toinduce precise cleavage at endogenous genomic loci in human and mouse cells.Cas9 can also be converted into a nicking enzyme to facilitatehomology-directed repair with minimal mutagenic activity. Finally, multipleguide sequences can be encoded into a single CRISPR array to enablesimultaneous editing of several sites within the mammalian genome,demonstrating easy programmability and wide applicability of the CRISPRtechnology. Each of the RNA segments can target a different sequence.
"That's the beauty of this—you can easily program a nucleaseto target one or more positions in the genome," Zhang says.
Although for this study, the researchers tested the systemin cells grown in the lab, they now plan to apply the new technology to studybrain function and diseases.
The new technique has broad application potential, saysZhang. The system could be used to design new therapies for diseases such asHuntington's disease, cystic fibrosis, autism, diabetes, neurodegenerativediseases—any medical condition caused by a genetic mutation. The system mightalso be useful for treating HIV by removing patients' lymphocytes and mutatingthe CCR5 receptor, through which the virus enters cells. After being put backin the patient, such cells would resist infection. And of course, as Zhangpoints out, the approach could also make it easier to study human disease byinducing specific mutations in human stem cells.
"Using this genome editing system, you can verysystematically put in individual mutations and differentiate the stem cellsinto neurons or cardiomyocytes and see how the mutations alter the biology ofthe cells," he says. "Anything that requires engineering of an organism to putin new genes or to modify what's in the genome will be able to benefit fromthis."
The study, "Multiplex Genome Engineering Using CRISPR/CasSystems," was published Jan. 3 in ScienceExpress, an electronic publication of the American Association for theAdvancement of Science journal Science.Lead authors of the paper are graduate students Le Cong and Ann Ran. Fundingcame from a variety of sources, including, notably, newscaster Jane Pauley, aswell as the U.S. National Institute of Mental Health; the W.M. Keck Foundation;the McKnight Foundation; the Bill & Melinda Gates Foundation; the DamonRunyon Cancer Research Foundation; the Searle Scholars Program; and MIT alumniMike Boylan and Bob Metcalfe.