Tag Archives: urine

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

(iStock)

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.

A pumpkin-shaped molecule for the first real-time methamphetamine and amphetamine sensor

A Sept. 28,2017 news item on Nanowerk announces a portable, inexpensive sensor for drugs (Note: A link has been renewed),

Speed, uppers, chalk, glass, crystal, or whatever you prefer to call them, can be instantly detected from biological fluids with a new portable kit that costs as little as $50. Scientists at the Center for Self-Assembly and Complexity, within the Institute for Basic Science (IBS, South Korea), in collaboration with Pohang University of Science and Technology (POSTECH), have devised the first methamphetamine and amphetamine sensor that can detect minute concentrations of these drugs from a single drop of urine in real-time.

Published in the journal Chem (“Point-of-Use Detection of Amphetamine-Type Stimulants with Host-Molecule-Functionalized Organic Transistors”), this simple and flexible sensor, which can be attached to a wristband and connected to an Android app via Bluetooth, could move drug screening from the labs to the streets.

A Sept. 28 (?), 2017 IBS press release by Letizia Diamante (also on EurekAlert), which originated the news item, expands on the theme,

Easy to synthesize and cheaper than heroin or cocaine, amphetamine-based drugs are the most abused drugs in the world, after cannabis. Conventional drug detection methods require a long time, as the sample must be taken into a lab for the analysis. It also needs experts to run the expensive equipment. The technology reported in this study is instead small, portable, cheap, fast and easy to use.

The idea for this technology came from the IBS chemist HWANG Ilha: “I was watching a TV news report on the usage of illegal drugs, and I thought to check what the chemical structure of methamphetamine looks like.” Soon after, the scientist anticipated that the drug would form a tight complex with a family of hollow pumpkin-shaped molecules, called cucurbituril (CB) members. The team then discovered that cucurbit[7]uril (CB[7])’s empty cavity binds well with amphetamine-based drugs and can be used as the drug recognition unit of a sensor. Cucurbiturils’ hollow chamber has already been studied for various technological uses, but this is the first device application in amphetamine-based drug detection.


▲ Figure 1: Wireless sensor for amphetamine-based drug detection.The kit is made of an organic field-effect transistor (OFET) device, an electric circuit board with a rechargeable battery and an antenna. The OFET device surface is coated with CB[7], whose function is to bind amphetamine and methamphetamine drugs in solution. The binding event is instantly converted to current, whose magnitude is proportional to the concentration of the drug. The app on the smartphone shows a peak as soon as a drop of urine with the drug is applied to the device. Moreover the entire kit can fit in a handy wristband.


▲ Video 1: The detector in action.
[Click text not image]
As soon as a drop of water with 0.0001 ng/mL (1 pM) of amphetamine is applied to the kit, the app shows a peak in current proportional to the concentration of drug. When the liquid is removed, the current level goes back to baseline, and the sensor can be reused. (Modified from Jang et al, Chem 2017)

Combining a transistor coated with CB[7], flexible materials, rechargeable batteries and a Bluetooth antenna, the research team developed a detector wristband connected to an app. In the presence of the drug, the molecular recognition between CB[7] and the drug molecule triggers an electrical signal which appears as a peak on the smartphone screen.

Current drug detection based on immunoassay or liquid chromatography/mass spectrometry techniques has a detection limit of about 10 ng/mL. On the contrary, the sensitivity of this new sensor is about 0.0001 ng/mL in water and 0.1 ng/mL in urine. Therefore, it is expected that this method will allow the detection of drug molecules in biological fluids, like urine and sweat, for a longer time after drug consumption.


▲ Figure 2: Graphic representation of the drug detection platform.Binding of drug molecules to the hollow cucurbit[7]uril (CB[7])’s cavity changes the current signal flowing in the transistor and therefore can be used as a detection system. The molecular structure of amphetamine and methamphetamine bound to cucurbit[7]uril (CB[7]) was confirmed with X-ray crystallography. Each color indicates a different atom (blue: nitrogen, red: oxygen, gray: carbon, and white: hydrogen). CB[7]’s hydrogen atoms have been omitted for clarity.


▲ Figure 3: Humorous view of the pumpkin-shaped molecule, cucurbit[7]uril (CB[7]), able to bind and detect amphetamine and methamphetamine molecules.(Credits: Modified from Titusurya – Freepik.com)

“Real time detection of amphetamine drugs on location would bring a big change to society,” explains another corresponding author KIM Kimoon. “In the same way as police can use a breathalyzer to detect alcohol on the spot, we aim to achieve the same with this device.”

False positives cannot be excluded yet, as urine contains a rich mixture of proteins and other metabolites that could affect the reading. Therefore, before commercializing it, clinical trials with drug users’ biological fluids are necessary. The researchers have patented the technology and they will continue to do further research in the near future.s

“Combining basic science with the latest technology, we can expect that this research will also lead to other new sensors, useful for our daily life,” concludes the third corresponding author OH Joon Hak. Indeed, the team is also keen on developing sensors for other kinds of drugs, as well as kits for the detection of dangerous substances, environmental monitoring, healthcare and safety.

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

Point-of-Use Detection of Amphetamine-Type Stimulants with Host-Molecule-Functionalized Organic Transistors by Yoonjung Jang, Moonjeong Jang, Hyoeun Kim, Sang Jin Lee, Eunyeong Jin, Jin Young Koo, In-Chul Hwang, Yonghwi Kim, Young Ho Ko, Ilha Hwang., Joon Hak Oh, Kimoon Kim. Chem (2017). DOI: 10.1016/j.chempr.2017.08.015 Publication stage: In Press Corrected Proof

This paper appears to be behind a paywall.