Tag Archives: polymerase chain reaction (PCR)

CRISPR-Cas12a as a new diagnostic tool

Similar to Cas9, Cas12a is has an added feature as noted in this February 15, 2018 news item on ScienceDaily,

Utilizing an unsuspected activity of the CRISPR-Cas12a protein, researchers created a simple diagnostic system called DETECTR to analyze cells, blood, saliva, urine and stool to detect genetic mutations, cancer and antibiotic resistance and also diagnose bacterial and viral infections. The scientists discovered that when Cas12a binds its double-stranded DNA target, it indiscriminately chews up all single-stranded DNA. They then created reporter molecules attached to single-stranded DNA to signal when Cas12a finds its target.

A February 15, 2018 University of California at Berkeley (UC Berkeley) news release by Robert Sanders and which originated the news item, provides more detail and history,

CRISPR-Cas12a, one of the DNA-cutting proteins revolutionizing biology today, has an unexpected side effect that makes it an ideal enzyme for simple, rapid and accurate disease diagnostics.

blood in test tube


Cas12a, discovered in 2015 and originally called Cpf1, is like the well-known Cas9 protein that UC Berkeley’s Jennifer Doudna and colleague Emmanuelle Charpentier turned into a powerful gene-editing tool in 2012.

CRISPR-Cas9 has supercharged biological research in a mere six years, speeding up exploration of the causes of disease and sparking many potential new therapies. Cas12a was a major addition to the gene-cutting toolbox, able to cut double-stranded DNA at places that Cas9 can’t, and, because it leaves ragged edges, perhaps easier to use when inserting a new gene at the DNA cut.

But co-first authors Janice Chen, Enbo Ma and Lucas Harrington in Doudna’s lab discovered that when Cas12a binds and cuts a targeted double-stranded DNA sequence, it unexpectedly unleashes indiscriminate cutting of all single-stranded DNA in a test tube.

Most of the DNA in a cell is in the form of a double-stranded helix, so this is not necessarily a problem for gene-editing applications. But it does allow researchers to use a single-stranded “reporter” molecule with the CRISPR-Cas12a protein, which produces an unambiguous fluorescent signal when Cas12a has found its target.

“We continue to be fascinated by the functions of bacterial CRISPR systems and how mechanistic understanding leads to opportunities for new technologies,” said Doudna, a professor of molecular and cell biology and of chemistry and a Howard Hughes Medical Institute investigator.

DETECTR diagnostics

The new DETECTR system based on CRISPR-Cas12a can analyze cells, blood, saliva, urine and stool to detect genetic mutations, cancer and antibiotic resistance as well as diagnose bacterial and viral infections. Target DNA is amplified by RPA to make it easier for Cas12a to find it and bind, unleashing indiscriminate cutting of single-stranded DNA, including DNA attached to a fluorescent marker (gold star) that tells researchers that Cas12a has found its target.

The UC Berkeley researchers, along with their colleagues at UC San Francisco, will publish their findings Feb. 15 [2018] via the journal Science’s fast-track service, First Release.

The researchers developed a diagnostic system they dubbed the DNA Endonuclease Targeted CRISPR Trans Reporter, or DETECTR, for quick and easy point-of-care detection of even small amounts of DNA in clinical samples. It involves adding all reagents in a single reaction: CRISPR-Cas12a and its RNA targeting sequence (guide RNA), fluorescent reporter molecule and an isothermal amplification system called recombinase polymerase amplification (RPA), which is similar to polymerase chain reaction (PCR). When warmed to body temperature, RPA rapidly multiplies the number of copies of the target DNA, boosting the chances Cas12a will find one of them, bind and unleash single-strand DNA cutting, resulting in a fluorescent readout.

The UC Berkeley researchers tested this strategy using patient samples containing human papilloma virus (HPV), in collaboration with Joel Palefsky’s lab at UC San Francisco. Using DETECTR, they were able to demonstrate accurate detection of the “high-risk” HPV types 16 and 18 in samples infected with many different HPV types.

“This protein works as a robust tool to detect DNA from a variety of sources,” Chen said. “We want to push the limits of the technology, which is potentially applicable in any point-of-care diagnostic situation where there is a DNA component, including cancer and infectious disease.”

The indiscriminate cutting of all single-stranded DNA, which the researchers discovered holds true for all related Cas12 molecules, but not Cas9, may have unwanted effects in genome editing applications, but more research is needed on this topic, Chen said. During the transcription of genes, for example, the cell briefly creates single strands of DNA that could accidentally be cut by Cas12a.

The activity of the Cas12 proteins is similar to that of another family of CRISPR enzymes, Cas13a, which chew up RNA after binding to a target RNA sequence. Various teams, including Doudna’s, are developing diagnostic tests using Cas13a that could, for example, detect the RNA genome of HIV.

infographic about DETECTR system

(Infographic by the Howard Hughes Medical Institute)

These new tools have been repurposed from their original role in microbes where they serve as adaptive immune systems to fend off viral infections. In these bacteria, Cas proteins store records of past infections and use these “memories” to identify harmful DNA during infections. Cas12a, the protein used in this study, then cuts the invading DNA, saving the bacteria from being taken over by the virus.

The chance discovery of Cas12a’s unusual behavior highlights the importance of basic research, Chen said, since it came from a basic curiosity about the mechanism Cas12a uses to cleave double-stranded DNA.

“It’s cool that, by going after the question of the cleavage mechanism of this protein, we uncovered what we think is a very powerful technology useful in an array of applications,” Chen said.

Here’s a link to and a citation for the paper,

CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity by Janice S. Chen, Enbo Ma, Lucas B. Harrington, Maria Da Costa, Xinran Tian, Joel M. Palefsky, Jennifer A. Doudna. Science 15 Feb 2018: eaar6245 DOI: 10.1126/science.aar6245

This paper is behind a paywall.

An easier, cheaper way to diagnose Ebola

A Sept. 9, 2015 news item on Nanotechnology Now highlights a new technology for diagnosing the Ebola virus,

A new Ebola test that uses magnetic nanoparticles could help curb the spread of the disease in western Africa. Research published in Biosensors and Bioelectronics shows that the new test is 100 times more sensitive than the current test, and easier to use. Because of this, the new test makes it easier and cheaper to diagnose cases, enabling healthcare workers to isolate patients and prevent the spread of Ebola.

The authors of the study, from the Chinese Academy of Sciences, say their new technology could be applied to the detection of any biological molecules, making it useful to diagnose other infectious diseases, like flu, and potentially detect tumors and even contamination in wastewater.

A Sept. 9, 2015 Elsevier press release, which originated the news item, provides more detail,

The Ebola virus causes an acute illness that is deadly in half of all cases, on average. The current outbreak of Ebola, which started in March 2014, affects countries in west Africa. In the most severely affected countries, like Guinea, Liberia and Sierra Leone, resources are limited, making control of the outbreak challenging. There is no vaccine for Ebola, so detecting the virus is key to controlling the outbreak: with an accurate diagnosis, patients can be isolated and treated properly, reducing the risk of spread.

“In west Africa, resources are under pressure, so complicated, expensive tests are not very helpful,” said Professor Xiyun Yan, one of the authors of the study from the Chinese Academy of Sciences. “Our new strip test is a simple, one-step test that is cheap and easy to use, and provides a visible signal, which means people don’t need training to use it. We think it will be especially helpful in rural areas, where technical equipment and skills are not available.”

Currently there are two ways to test for the Ebola virus: using a method called polymerase chain reaction (PCR), which makes copies of the molecules for detection, and with antibody-capture enzyme-linked immunosorbent assay (ELISA), which gives a visual indication when a given molecule is in a sample. PCR is very sensitive, but is expensive and complicated, requiring special skills and technical equipment. The ELISA – or gold strip test – is cheaper but sensitivity is very low, which means it often gives the wrong results.

The new test, called the nanozyme test, uses magnetic nanoparticles, which work like enzymes to make the signal stronger, giving a clearer result you can see with the naked eye. The test can detect much smaller amounts of the virus, and is 100 times more sensitive than the gold strip test.

“People have loved the strip test for many years, but it has a major weakness: it’s not sensitive enough. We’re very excited about our new nanozyme test, as it is much more sensitive and you don’t need any specialist equipment to get a quick, accurate result,” said Dr. Yan.

Strip tests work by attaching molecules called antibodies to gold particles to look for a particular molecule in a sample. When they attach to the molecule you’re looking for, in this case a virus, they produce a signal, such as a color change. In order to find the virus, the particles need to be labelled with enzymes, which speed up detection and signalling.

With the new nanozyme test, the researchers applied magnetic nanoparticles as a nanozyme probe in place of gold nanoparticles. After labeling with an antibody that attaches to the Ebola virus, this novel probe is able to recognize and separate the virus in a sample. The nanoparticles are magnetic, so to concentrate the virus particles in a sample, all you need to do is hold the sample against a magnet; no expensive equipment is needed.

The nanozyme test is 100 times more sensitive than the gold strip test, detecting molecules called glycoproteins on the surface of the Ebola virus at concentrations as low as 1 nanogram per milliliter.

The researchers have applied for a patent for the new test, which is currently being taken to west Africa by the CDC to use in the field. The researchers are also collaborating with clinical teams to apply the technology to other diseases, and with a company that treats wastewater to see if it can help remove environmental contamination.

Here’s a link to and a citation for the paper,

Nanozyme-strip for rapid local diagnosis of Ebola by Demin Duan, Kelong Fan, Dexi Zhang, Shuguang Tan, Mifang Liang, Yang Liu, Jianlin Zhang, Panhe Zhang, Wei Liu, Xiangguo Qiu, Gary P. Kobinger, George Fu Gao, Xiyun Yan. Biosensors and Bioelectronics Volume 74, 15 December 2015, Pages 134–141 doi:10.1016/j.bios.2015.05.025

This paper appears to be open access.