CRISPR-based DNA detective

Researchers have developed a CRISPR-based DNA-detection system, DETECTR, that could lead to fast, reliable medical tests.

The technology, developed in the laboratory of Howard Hughes Medical Institute (HHMI) Investigator Jennifer Doudna at the University of California, Berkeley, can spot snippets of DNA that signal viral infections, cancer or even defective genes. The genetic detective was created by combining the capabilities of CRISPR with a molecular flare gun.

The new method “enables sensitive and accurate DNA detection”, Doudna said, and was able to spot two types of cancer-causing human papillomavirus, or HPV, in human samples. The results were published in the journal Science.

DETECTR, or DNA Endonuclease Targeted CRISPR Trans Reporter, is based on CRISPR, a bacterial defence system that cuts and disables viruses and other threats. Since its discovery, CRISPR has been co-opted by scientists eager to selectively snip and repair genomes. In the new study, Doudna and her colleagues show that CRISPR biology can be used in another way, too — as a DNA homing beacon.

The new technology relies on Cas12a, an enzyme described in 2015. Like its molecular cousin Cas9, which Doudna and colleague Emmanuelle Charpentier turned into a genome editing tool in 2012, Cas12a snips DNA. But instead of snipping only the DNA strand it binds, Cas12a chops other DNA, too. “We started to see this surprising activity where it would just start cutting random stuff,” said study co-author Lucas Harrington, a graduate student in Doudna’s lab.

Under certain circumstances, the enzyme turns into a DNA shredding machine, slicing up any single-stranded DNA nearby, the researchers saw. But this wasn’t indiscriminate destruction. For the machete action to begin, Cas12a first has to find a precise DNA target. Researchers can program that target by adding a guide — an RNA molecule that tells Cas12a what to look for. “It’s so easy to reprogram this to find any piece of DNA that you want to detect,” Harrington said.

Once Cas12a locks onto and snips the target, it then begins shredding all of the single-stranded DNA it can find. But for the system to be useful, Doudna and colleagues needed a way to see when Cas12a starts this molecular mayhem, signalling that it has found its target. So the researchers used a glowing molecule — an easy-to-spot flare — linked by a single strand of DNA to a suppressor molecule that prevents the glow. When Cas12a turns into a machete, it slices the DNA strand that links these two molecules together. This removes the suppressor, letting the glowing molecule shine — a signal researchers can detect.

Infographic by HHMI.

The team then put their DNA detective to the test. Working with Dr Joel Palefsky and his team at the University of California, San Francisco, they hunted for DNA signals from two types of cancer-causing HPV: type 16 and type 18. Researchers obtained 25 DNA samples taken from people who had no HPV infection, one type of virus or both types. For HPV16, DETECTR made the right call for all 25 samples. For HPV18, DETECTR got it right for 23 of 25 samples. The ones it missed gave weak signals that can likely be improved with different guide RNA design, Doudna said.

Compared with current methods to detect HPV, DETECTR is “simpler, quicker, and does not require specialised equipment”, Harrington said. That could make the system useful in resource-limited health clinics and for point-of-care diagnostics.

Doudna’s team’s method could easily be applied to other types of viral or bacterial infections, and even cancer markers, chromosomal abnormalities or other genetic signals, Harrington said. More generally, the results highlight the promise of basic biology. Basic research on an ancient bacterial defence system keeps turning up new surprises, and new potential uses. CRISPR “is a treasure chest that we keep digging into and finding new things”, Harrington said.

Janice S. Chen et al., CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. Published online February 15, 2018. doi: 10.1126/science.aar6245

Top image caption: HHMI Investigator Jennifer Doudna and colleagues have created DETECTR. Credit: Ryan Anson.