Bacteria have a secret weapon for dealing with viral invaders: a library of genetic mug shots. These bits of DNA, collected from previously encountered viruses, help the bacteria target and destroy their invaders. HHMI scientists recently showed that this defense mechanism—known as the CRISPR-Cas system—gets some of its accuracy and speed from a tiny sequence of DNA that is just three nucleotides long.
The workhorse of the CRISPR-Cas immune system is an enzyme called Cas9. Each Cas9 molecule carries a 20-base pair “guide” RNA sequence that matches one of the DNA mug shots. When a repeat offender invades, it’s up to the Cas9 complex to find the complementary DNA sequence on the pathogen and to cut it.
Scientists also use the CRISPR system in their research labs to make precise changes in the genomes of animals and plants. “One of the concerns for people who are using this as a tool has been whether there are off-target effects, in which sites are mistakenly recognized by Cas9 and perhaps cut or modified in experiments,” says HHMI Investigator Jennifer Doudna from the University of California, Berkeley.
Doudna teamed up with HHMI Early Career Scientist Eric Greene of Columbia University to figure out how Cas9 does its job. Using a technique called a DNA curtains assay that was pioneered by Greene, the scientists were able to watch Cas9 interact with individual DNA molecules. Their findings were published on March 6, 2014, in Nature.
Instead of inspecting the entire genome of an invading virus, Cas9 bounces onto and off of the viral DNA while looking for a specific three-letter sequence of nucleotides, called a PAM. “The enzyme really only spends time at sites that have this motif,” explains Doudna.
|Cas9: The Enzyme, The RNA, & The Virus|
Cas9 unzips the DNA at each PAM site, looking for sequences that match its own guide RNA. There may be thousands of PAM sites, but only one sits next to the DNA pattern that Cas9 is looking for. If there’s no match, Cas9 falls off, and the search continues.
Knowing that Cas9 relies on a PAM sequence in addition to its RNA guide molecule will help reduce concerns about the technique and will guide efforts to make Cas9 a better tool in genome engineering.