A 3D representation of Cas9, the part of CRISPR/Cas9 that changes the DNA. Created with a 3D printer. (NIH Image Gallery)
CRISPR/Cas9 has gotten so much attention lately that even a pop culture hit like "The X-Files" has taken note. The revolutionary DNA-editing tool is appreciably simpler and more versatile than the techniques scientists had previouslyused to alter unwanted DNA.
And now a new process devised by researchers at Dr. David Liu's lab at Harvard University, described in the journal Nature last week, appears to make CRISPR/Cas9 more efficient at fixing DNA while causing less collateral damage to boot.
While this new version cannot fix as many broken genes as the original, on balance it appears to be a better-behaved genome editing tool, potentially giving scientists a real chance to cure certain genetic diseases.
The new technique is so much more precise, you can think of it this way: Where the old system is the equivalent of correcting a single spelling error by copying and pasting a whole new section that includes the right letter, this new technique enables you to make the correction by simply deleting the incorrect letter and substituting the right one.
CRISPR/Cas9 will almost certainly transform medicine, as well, giving scientists the ability to treat or cure genetic diseases through the repair of broken genes. Scientists have even tentatively begun to tweak DNA in human embryos as a first step toward curing severe genetic illnesses before they occur.
The Old Way
CRISPR/Cas9 edits genes by using three components.
RNA, a close relative to DNA, is used as a precise targeting device to home in on a gene that needs correcting. Cas9, an enzyme, travels with the RNA and makes a cut in the DNA at a specific, problematic spot. New, added DNA that has the corrected sequence -- the third component -- is then used by the cell's internal machinery to correct the gene.
A key strength of this technique is its ability to send Cas9 where it should and nowhere else -- most of the time. But its efficiency in editing, however, is not as topnotch. Usually only a few cells end up with the desired change, so that in many cases no effect can be seen.
Even more problematic is that more often than not, after Cas9 cuts the original DNA, the cell -- in a sort of panic -- will immediately try to fill the gap, adding to or subtracting from the gene's code, potentially damaging the DNA further.
Now! New and Improved!
To solve this problem, the Harvard researchers created two radically changed versions of Cas9, which they called BE2 and BE3. Both are much better at changing the DNA and less likely to damage it.
The scientists started by using a form of Cas9 that could be directed to the right place in the genome but could not cut DNA. To this inactive Cas9 they added an enzyme (cytidine deaminase). This changes an unwanted C -- a molecule called cytosine that is one of the four bases found in someone's genetic code -- into a U, a base found in RNA that isvery similar to a T (thymine), another DNA base.
They called this new version BE1. Essentially, BE1 changes Cs to Ts without an incision -- and the resulting damage -- in the DNA.
But although BE1 worked very well in a test tube, it didn't perform as well in a cell. That's because cells frequently like to replace the newly added U with the old C. (Click here for why the cell has such a system.)
The researchers fixed this problem by kludging onto BE1 something from bacteria called uracil DNA glycosylase inhibitor (UGI), which makes it more difficult for the cell to put back the C. This new version, which still does not cut the DNA, was called BE2.
In a final step to make an even better tool, they tweaked Cas9 one last time, partially restoring its ability to cut DNA. However, in this version, Cas9 was engineered to cut only a single strand of DNA, opposite the C. Because cells have a much more precise system for this type of repair, less damage is done. This version was called BE3.
This new technique is fundamentally different from the old one. Instead of cutting the DNA and relying on the cell's machinery to repair a gene, BE2 and BE3 actually go in and swap out a single letter of DNA.
You can see the advantages of BE2 and BE3 over the old Cas9 in the following results obtained after editing a particular DNA site:
% Cells with a Fixed Gene
% Cells with a Damaged Gene
% Cells with an Unaffected Gene
Less than 0.1
Similarly, improved efficiency was seen at 14 other locations in the DNA of six different genes, with the number of cells repaired hitting a high of 75 percent.
These improvements are significant enough that the new versions might be able to cure a disease whereas the old Cas9 might fall short.
If these results can be repeated at other sites, it looks like the lab will have built a better CRISPR mousetrap.
The disadvantage of the new technique is that unlike old-school Cas9, neither BE2 nor BE3 will work on every gene or piece of DNA. Only those genes with mutations in which a C has been changed to a T can be fixed.
Because DNA is made up of four bases -- A and G in addition to C and T -- the inability to repair more than one permutation is definitely limiting. But it still means a lot of repaired genes. The authors, in fact, compiled a list of 300-900 mutations in genes that cause diseases like cystic fibrosis and Leigh disease and can be fixed with these new versions of Cas9.
Videos: How CRISPR works
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