Editing the genome: New method allows scientists to insert multiple genes in specific locations, delete defective genes

Jan 03, 2013 by Anne Trafton
A new technique developed at MIT can edit DNA in precise locations. Graphic: Christine Daniloff/iMol

Researchers at 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. The researchers say the technology could offer an easy-to-use, less-expensive way to engineer organisms that produce biofuels; to design animal models to study human disease; and to develop new therapies, among other potential applications.

To create their new genome-editing technique, the researchers modified a set of that normally defend against viral invaders. Using this system, scientists can alter several genome sites simultaneously and can achieve much greater control over where new are inserted, says Feng Zhang, an assistant professor of brain and cognitive sciences at MIT and leader of the research team.

"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," says Zhang, who is a core member of the Broad Institute and MIT's McGovern Institute for .

Zhang and his colleagues describe the new technique in the Jan. 3 online edition of Science. Lead authors of the paper are graduate students Le Cong and Ann Ran.

Early efforts

The first genetically altered mice were created in the 1970s by adding small pieces of DNA to mouse . This method is now widely used to create for the study of , but, because it inserts DNA randomly in the genome, researchers can't target the newly delivered genes to replace existing ones.

In recent years, scientists have sought more precise ways to edit the genome. One such method, known as homologous , 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.

Precise targeting

The new system is much more user-friendly, Zhang says. Making use of naturally occurring bacterial protein-RNA systems that recognize and snip viral DNA, the researchers can create 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.

This approach can be used either to disrupt the function of a gene or to replace it with a new one. To replace the gene, the researchers must also add a DNA template for the new gene, which would be copied into the genome after the DNA is cut.

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.

The method is also very precise—if there is a single base-pair difference between the RNA targeting sequence and the genome sequence, Cas9 is not activated. This is not the case for zinc fingers or TALEN. The new system also appears to be more efficient than TALEN, and much less expensive.

The research team has deposited the necessary genetic components with a nonprofit called Addgene, making the components widely available to other researchers who want to use the system. The researchers have also created a website with tips and tools for using this new technique.

Engineering new therapies

Among other possible applications, this system could be used to design new therapies for diseases such as Huntington's disease, which appears to be caused by a single abnormal gene. Clinical trials that use zinc finger nucleases to cut out the gene and replace it with the normal version are now under way, and the new technology could offer a more efficient alternative.

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.

This approach could also make it easier to study human disease by inducing specific mutations in human stem cells. "Using this 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," Zhang says.

In the Science study, the researchers tested the system in cells grown in the lab, but they plan to apply the new technology to study brain function and diseases.

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Sean_W
1 / 5 (2) Jan 03, 2013
I thought there were already researchers who had combined this method with self assembling virus parts to create a complex which does all that is described here AND can be injected into an organism to change specific cells without removal, cloning and reinsertion. I recall them saying they had cured a specific genetically derived bleeding disorder in mice using that method. They effectively injected the fake viruses into the mouse, they went into the liver cells and replaced the damaged gene.

I'm glad that other people are working on this but this story seems a bit less impressive as there is the added steps of acquiring and returning the patient's cells.
PPihkala
not rated yet Jan 03, 2013
What I have been wondering what would be the benefit of fixing the damaged C vitamin manufacturing pathway in humans. This tech should allow that mod also.
Sean_W
1 / 5 (2) Jan 03, 2013
What I have been wondering what would be the benefit of fixing the damaged C vitamin manufacturing pathway in humans. This tech should allow that mod also.


It might be beneficial for people living in the high arctic, far from distribution centres. But then multivitamins might remain cheaper than gene swapping for a while. But that and other sites might make convenient locations for other genes like the kind that give crocodiles the brain protein which stores oxygen for long swims between breaths. Or we might just swap out our version of myoglobin for the one that whales have that lets them store huge amounts of oxygen in their muscles.
C_elegans
not rated yet Jan 03, 2013
The authors report 10-25% effeciency in hek293 cells, that's 100-1000x better than any previous method.
extinct
1 / 5 (5) Jan 04, 2013
genetic modification is the first step in the extinction of humans as we know them. when we are no longer natural the way nature and evolution made us, then we are no longer ourselves. when a laboratory made part of you, you are their slave thereafter. when our genes are patented and owned by Monsanto or someone else for profit, that's the end. at current rates, I predict it will come within 2 or 3 centuries.
Shabs42
not rated yet Jan 04, 2013
when a laboratory made part of you, you are their slave thereafter.


Not unless they program some sort of self destruct into your genes. With your logic you could argue that a significant portion of us are already slaves because we've taken life saving prescription medications that were made in a lab. At the very least you're saying test tube babies are slaves and as far as I know they have just as much free will as the rest of us.

I do agree with you that naturally occurring genes should not be patentable though, that's BS.
C_elegans
not rated yet Jan 04, 2013
when we are no longer natural the way nature and evolution made us, then we are no longer ourselves.


So? Does life end or do we somehow carry on?

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