The most vital gift your mom and father ever gave you was once the two units of three billion letters of DNA that make up your genome. But like some thing with three billion components, that gift is fragile. Sunlight, smoking, unhealthy eating, even spontaneous errors made by way of your cells, all reason changes to your genome.
The most frequent form of change in DNA is the easy swap of one letter, or base, such as C, with a different letter, such as T, G or A. In any day, the cells in your body will collectively accumulate billions of these single-letter swaps, which are additionally called “point mutations.” Now, most of these point mutations are harmless.
But every now and then, a factor mutation disrupts an vital functionality in a cell or causes a cell to misbehave in damaging ways. If that mutation have been inherited from your mother and father or occurred early enough in your development, then the end result would be that many or all of your cells contain this harmful mutation.
And then you would be one of lots of hundreds of thousands of human beings with a genetic disease, such as sickle cell anemia or progeria or muscular dystrophy or Tay-Sachs disease. Grievous genetic illnesses caused by point mutations are particularly frustrating, because we regularly know the exact single-letter change that causes the disorder and, in theory, could cure the disease. Millions go through from sickle cell anemia due to the fact they have a single A to T point mutations in both copies of their hemoglobin gene.
And young people with progeria are born with a T at a single position in their genome where you have a C, with the devastating consequence that these wonderful, bright kids age very swiftly and pass away by about age 14. Throughout the history of medicine, we have not had a way to successfully correct point mutations in living systems, to change that disease-causing T back into a C. Perhaps till now.
Because my laboratory recently succeeded in creating such a capability, which we call “base editing.” The story of how we developed base enhancing actually begins three billion years ago. We think of micro organism as sources of infection, however micro organism themselves are also prone to being infected, in particular, by viruses. So about three billion years ago, micro organism evolved a defense mechanism to fight viral infection. That protection mechanism is now better recognized as CRISPR.
And the warhead in CRISPR is this purple protein that acts like molecular scissors to cut DNA, breaking the double helix into two pieces. If CRISPR could not distinguish between bacterial and viral DNA, it would not be a very beneficial defense system. But the most terrific feature of CRISPR is that the scissors can be programmed to search for, bind to and cut only a unique DNA sequence. So when a bacterium encounters a virus for the first time, it can keep a small snippet of that virus’s DNA for use as a program to direct the CRISPR scissors to cut that viral DNA sequence during a future infection. Cutting a virus’s DNA messes up the function of the cut viral gene, and consequently disrupts the virus’s lifestyles cycle.
Remarkable researchers together with Emmanuelle Charpentier, George Church, Jennifer Doudna and Feng Zhang confirmed six years ago how CRISPR scissors could be programmed to cut DNA sequences of our choosing, which include sequences in your genome, as an alternative of the viral DNA sequences chosen by bacteria.
But the effects are actually similar. Cutting a DNA sequence in your genome additionally disrupts the function of the cut gene, typically, through causing the insertion and deletion of random mixtures of DNA letters at the cut site. Now, disrupting genes can be very beneficial for some applications. But for most point mutations that cause genetic diseases, virtually cutting the already-mutated gene won’t benefit patients, due to the fact the function of the mutated gene needs to be restored, not further disrupted. So cutting this already-mutated hemoglobin gene that causes sickle cell anemia won’t restore the ability of patients to make healthy red blood cells.
And whilst we can once in a while introduce new DNA sequences into cells to change the DNA sequences surrounding a cut site, that process, unfortunately, does not work in most types of cells, and the disrupted gene effects still predominate. Like many scientists, I’ve dreamed of a future in which we would possibly be capable to deal with or perhaps even cure human genetic diseases. But I noticed the lack of a way to fix point mutations, which cause most human genetic diseases, as a major trouble standing in the way.
Being a chemist, I began working with my college students to boost methods on performing chemistry at once on an individual DNA base, to truly fix, rather than disrupt, the mutations that cause genetic diseases. The consequences of our efforts are molecular machines called “base editors.” Base editors use the programmable searching mechanism of CRISPR scissors, however instead of cutting the DNA, they directly convert one base to another base without disrupting the rest of the gene. So if you think of naturally occurring CRISPR proteins as molecular scissors, you can think of base editors as pencils, capable of immediately rewriting one DNA letter into another through actually rearranging the atoms of one DNA base to instead turn out to be a different base.
Now, base editors do not exist in nature. In fact, we engineered the first base editor, shown here, from three separate proteins that do not even come from the same organism. We began with the aid of taking CRISPR scissors and disabling the ability to cut DNA whilst retaining its ability to search for and bind a target DNA sequence in a programmed manner. To these disabled CRISPR scissors, shown in blue, we attached a 2d protein in red, which performs a chemical reaction on the DNA base C, changing it into a base that behaves like T. Third, we had to connect to the first two proteins the protein shown in purple, which protects the edited base from being removed by the cell.
The net end result is an engineered three-part protein that for the first time allows us to convert Cs into Ts at specified places in the genome. But even at this point, our work was only 1/2 done. Because in order to be stable in cells, the two strands of a DNA double helix have to form base pairs. And due to the fact C only pairs with G, and T only pairs with A, simply altering a C to a T on one DNA strand creates a mismatch, a disagreement between the two DNA strands that the cell has to resolve by deciding which strand to replace. We realized that we ought to further engineer this three-part protein to flag the nonedited strand as the one to be replaced via nicking that strand. This little nick tricks the cell into changing the nonedited G with an A as it remakes the nicked strand, thereby finishing the conversion of what used to be a C-G base pair into a stable T-A base pair.
After numerous years of hard work led by a former post doc in the lab, Alexis Komor, we succeeded in growing this first class of base editor, which converts Cs into Ts and Gs into As at targeted positions of our choosing. Among the more than 35,000 known disease-associated point mutations, the two types of mutations that this first base editor can reverse collectively account for about 14 percent or 5,000 or so pathogenic point mutations. But correcting the biggest fraction of disease-causing point mutations would require developing a 2d type of base editor, one that may want to convert As into Gs or Ts into Cs. Led by Nicole Gaudelli, a former post doc in the lab, we set out to increase this 2nd class of base editor, which, in theory, should correct up to nearly 1/2 of pathogenic point mutations, which include that mutation that causes the rapid-aging disease progeria. We realized that we could borrow, once again, the targeting mechanism of CRISPR scissors to bring the new base editor to the proper site in a genome.
But we shortly encountered an incredible problem; namely, there is no protein that’s known to convert A into G or T into C in DNA. Faced with such a serious stumbling block, most students would probably look for some other project, if not another research advisor. (Laughter) But Nicole agreed to proceed with a design that appeared wildly formidable at the time. Given the absence of a naturally going on protein that performs the imperative chemistry, we determined we would evolve our personal protein in the laboratory to convert A into a base that behaves like G, beginning from a protein that performs associated chemistry on RNA.
We set up a Darwinian survival-of-the-fittest resolution machine that explored tens of hundreds of thousands of protein versions and solely allowed those uncommon variations that ought to function the vital chemistry to survive. We ended up with a protein proven here, the first that can convert A in DNA into a base that resembles G. And when we connected that protein to the disabled C