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New CRISPR device detects genetic mutations in minutes



Kiana Aran's novel system immobilizes the CRISPR complexes on the surface of graphene-based transistors. These complexes look for a genome for their target sequence and bind to its DNA in a successful search. This bond changes the conductivity of the graphene material in the transistor, whereby the change is detected with a handheld reader. Picture credits: Keck Graduate Institute (KGI)

A team of engineers from the University of California, Berkeley and The Keck Graduate Institute (KGI) of The Claremont Colleges combined CRISPR with graphene electronic transistors to create a new handheld device that detects specific genetic mutations in a can be matter of minutes.

The CRISPR device could be used to quickly diagnose genetic disorders or to assess the accuracy of gene editing techniques. The team used the device to identify genetic mutations in DNA samples from Duchenne muscular dystrophy patients.

"We have developed the first transistor that uses CRISPR to search your genome for possible mutations," said Kiana Aran, assistant professor at KGI, who designed the technology during a postdoctoral fellowship at Irina Conboys Laboratory of Biotechnology at Berkeley University , "You simply put your cleaned DNA sample on the chip, allow CRISPR to search, and the graphene transistor reports the result of that search in minutes."

Aran is the lead author of a paper describing the device that appears online in March 25 in the journal Nature Biomedical Engineering .

Physicians and geneticists can now sequence DNA to identify genetic mutations that underlie a variety of traits and conditions, and companies such as 23 and Mee and AncestryDNA provide these tests to even curious consumers

However, in most forms of genetic testing, including the recently developed CRISPR-based diagnostic techniques, the CRISPR chip uses nanoelectronics to detect genetic mutations in DNA samples without previously "amplifying" or replicating the DNA segment of interest millions of times time- and device-intensive process called polymerase chain reaction or PCR. That is, it could be used to conduct genetic testing in a doctor's office or in field workplaces without having to send a sample to a laboratory.

"CRISPR chip has the advantage that it really is a point of care of the few things that you could really do it at the bedside if you had a good DNA sample," said Niren Murthy, a professor of Bioengineering at UC Berkeley and co-author of the newspaper. "Ultimately, all you have to do is remove one person's cells, extract the DNA and mix with the CRISPR chip, and you can tell if there is a specific DNA sequence or not, which could potentially lead to a true bedside assay DNA.

Bypassing the Bottleneck

The CRISPR-Cas9 system is known for its ability to cut DNA threads in precise locations like razor-sharp scissors, giving researchers unprecedented editing capabilities. For the Cas9 protein to accurately cut and insert genes, it must first determine the exact locations in the DNA that it needs to cut.

In order for Cas9 to find a specific location in the genome, scientists first have to use a section of "guide RNA," a small piece of RNA whose bases are complementary to the DNA sequence of interest. The bulky protein first opens the double-stranded DNA and scans it until it finds the sequence that corresponds to the lead RNA and then locks in.

To capitalize on CRISPR's ability to target genes, researchers used a deactivated Cas9 protein-a variant of Cas9 that can find a specific location on DNA but does not cut it-and attached to graphene transistors Has. When the CRISPR complex finds the location on the DNA it is targeting, it binds to it and triggers a change in the electrical conductivity of the graphene, which in turn alters the electrical properties of the transistor. These changes can be detected with a handheld device developed by the team's industrial staff.

Graphene, which consists of a single atomic carbon atom layer, is so electrically sensitive that it can detect a DNA sequence hit in a complete cell. Genome sample without PCR amplification.

To capitalize on CRISPR's ability to gene target, researchers took a deactivated Cas9 protein – a variant of Cas9 that finds a specific location on DNA but do not cut it off – and tie it to graphene transistors. When the CRISPR complex finds the location on the DNA it is targeting, it binds to it and triggers a change in the electrical conductivity of the graphene, which in turn alters the electrical properties of the transistor. Picture credits: Keck Graduate Institute

"Graphene's hypersensitivity enabled us to recognize CRISPR's DNA discovery activity," said Aran. "CRISPR brought the selectivity, graphene transistors brought the sensitivity and together we could perform this PCR-free or amplification-free detection."

Aran hopes to "multiplex" the device soon so that doctors can connect multiple devices Run the RNAs simultaneously to detect multiple genetic mutations within minutes.

"Imagine a page with many search fields, in our case transistors, and you have your lead RNA information in those search fields, and each of these transistors will search and report the result electronically," Aran said ,

Rapid Diagnostics

To demonstrate the sensitivity of the CRISPR chip, the team used the device to detect two common genetic mutations in blood samples from Duchenne patients with muscular dystrophy (DMD).

Conboy, co-author of the paper, says CRISPR could be a particularly useful tool for DMD screening, as the severe muscle-displacing disease can be caused by mutations in the whole mass. The active dystrophin gene – one of the longest in the human genome – and mutations in the speculation process can be costly and time-consuming with PCR-based genetic testing.

"Boys who suffer from DMD are usually unaware that something is wrong, and then undergo genetic confirmation," said Conboy, who also works on CRISPR-based treatments for DMD.

"With a digital device, you could design guide RNAs across the entire dystrophin gene, and then you could easily check the entire sequence of the gene in a matter of hours.You could alert parents or even newborns to the presence or absence of dystrophin If the mutation is found, therapy could start early before the disease actually develops, "Conboy said.

Murthy said doctors could also help develop individualized treatment plans. For example, genetic variations make some people unresponsive to expensive blood thinners like Plavix.

"If you have specific mutations or certain DNA sequences, it will be very accurate in predicting how you will respond to certain drugs," Murthy said.

Because the CRISPR chip can be used to monitor whether CRISPR binds to specific DNA sequences, it could also test the effectiveness of CRISPR-based gene editing techniques. For example, they could be used to verify the correct design of leader RNA sequences.

"Combining modern nanoelectronics with modern biology opens a new door to accessing new biological information that was previously inaccessible," said Aran.


Explore Further:
New strategy improves the efficiency of processing CRISPR Cas9 genomes

Further information:
Detection of unamplified target genes via CRISPR-Cas9 immobilized on a graphene field effect transistor Nature Biomedical Engineering (2019). DOI: 10.1038 / s41551-019-0371-x, https://www.nature.com/articles/s41551-019-0371-x

Magazine Reference:
Nature Biomedical Engineering

Provided by:
University of California, Berkeley


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