For all the power it has demonstrated, scientists are still not too sure how error-prone CRISPR-Cas9, the genome editing technique, might be. However, two new studies done by one of the pioneers of the technique may put these worries to bed.
CRISPR-Cas9 is composed of two integral parts: two strands of RNA called guide RNA, and an enzyme that can cut DNA known as Cas9.
The guide RNA can be engineered to match any sequence in a genome that is of interest to scientists. When the guide RNA binds to the genome, the Cas9 protein cuts the DNA. The next step depends on the goals of the project. Researchers can use the cut area to insert novel genes, or they can let the cell repair the cut itself a process that is quite sloppy and usually leads to the gene becoming inactive (i.e. knocked-out).
As scientists move from preforming this technique in their labs, to preforming it on humans, some concerns about the system have arisen. For example, scientists worry about the possibility that the guide RNA may attach to places in the genome that are a close match but not a 100 percent match. There are also concerns that Cas9 may begin making random cuts in the genome. These concerns are not minor, however, the new studies show that the system actually has three different mechanisms to ensure an elite level of accuracy and precision.
The studies, one published in Science and the other in Nature, come from the lab of Dr. Jennifer Doudna, one of the pioneers of CRISPR-Cas9. The Science study describes how the Doudna Lab, working at University of California at Berkeley, were able to observe as the system scanned billions of pieces of DNA to find exactly the target sequence to cut.
The researchers also noted that when the guide RNA did bind non-specifically, the Cas9 protein disengaged from the DNA almost immediately. In the the Nature paper, researchers noted that when the guide RNA does bind correctly, only then does the Cas9 protein undergo specific changes to its structure (known as conformational changes), which enable it to cut the DNA. The part of the Cas9 enzyme that cuts the DNA is inhibited until the guide RNA binds specifically.
These discoveries are vital to the progress of this technique, and are needed before it can be used in humans, an event that some say may happen as soon as 2017. But in truth, discovering that the system has these specific check points in place is not really a surprise.
CRISPR-Cas9 was not created in a lab, but discovered in bacteria. In these single-celled organisms, the CRISPR-Cas9 system works similar to how the immune system works in humans. The guide RNA the bacteria make are sequences that recognize portions of the genome of viruses that commonly invade them. When the guide RNA detects the virus, the Cas9 protein chops up the viral genome, thus inhibiting the infection.
If the system worked too non-specifically, then the guide RNA could bind to portions of the bacteria's own genome and the Cas9 protein would self-attack the bacteria. The system is fairly widespread among bacteria and Archaea and has evolved over billions of years. During this time, natural selection has favored versions of the system that simply do not make non-specific cuts.