Rapid genetic testing
Researchers at the University of California at Berkeley, working under the guidance of associate Professor Kiana Aran, have combined CRISPR genome editing technology with graphene electronic transistors to create a new portable device that allows identifying specific genetic mutations within a few minutes. The new device, called the CRISPR chip, can be used to quickly diagnose genetic diseases, as well as to assess the accuracy of gene editing methods.
Today, doctors and geneticists can use DNA sequencing to identify genetic mutations that cause various signs and conditions, and commercial companies such as 23andMe make such tests available to curious consumers.
However, unlike most methods of genetic testing, including recently developed diagnostic methods based on CRISPR technology, the CRISPR chip uses nanoelectronics to detect mutations in DNA samples without prior amplification – copying the DNA segment of interest several million times using a lengthy and demanding complex equipment process known as polymerase chain reaction (PCR). This means that the new method will allow genetic testing to be carried out in a doctor's office or in the "field", eliminating the need to deliver a sample to the laboratory.
The CRISPR chip uses a deactivated CRISPR-Cas9 protein attached to a graphene transistor to identify specific genetic sequences in a DNA sample.
The CRISPR-Cas9 system is known for its ability to cut DNA strands in precisely specified locations like a pair of razor-sharp scissors, providing researchers with unprecedented opportunities in gene editing. However, in order for the Cas9 protein to carry out the manipulation given to it, it first needs to accurately determine the place of DNA cutting. To do this, the researchers load it with "guide RNA" – a small fragment of RNA, the nucleic bases of which are complementary to the DNA sequence they are interested in. The clumsy protein first unwinds a double-stranded DNA molecule and scans it until it finds a sequence corresponding to the guide RNA, after which it attaches to it.
To take advantage of the CRISPR system's search capability, the researchers took a deactivated Cas9 protein capable of finding a specific region of DNA, but unable to cut it, and attached it to the surface of a graphene transistor. When bound to its target, the CRISPR complex starts the process of changing the electrical conductivity of graphene, which, in turn, changes the electrical characteristics of the transistor. These changes can be recorded using a specially designed portable device.
Graphene, made from a monatomic carbon layer, has such a high sensitivity to changes in electrical conductivity that it allows detecting a certain point on the DNA chain in a genome-wide sample, without DNA amplification using PCR.
The authors hope that in the near future they will be able to improve the technology by using several guide RNAs to detect different mutations.
To confirm the sensitivity of the CRISPR chip, the researchers successfully used it to identify two mutations in the DNA of blood cells of patients with Duchenne myodystrophy that most often cause this disease.
According to the authors, the CRISPR chip can be especially useful for screening for Duchenne myodystrophy, since this severe degenerative disease can be caused by numerous mutations in different parts of the dystrophin protein gene, which is one of the longest in the entire human genome. Detection of these mutations by PCR is a task that requires a lot of financial and time costs. When using a new digital device, it will take only a few hours to analyze the entire gene.
To date, boys with this rare disease (it occurs in about one in 5,000) undergo genetic testing only after doctors suspect the disease. The introduction of CRISPR-chip technology into clinical practice will allow screening of parents or newborns for the corresponding mutations, which will ensure the possibility of early initiation of therapy.
Cas9 protein molecules equipped with different RNA guides can be applied to each individual chip, which will allow for the simultaneous identification of different DNA sequences.
Rapid genetic testing will also allow doctors to personalize therapeutic protocols depending on the characteristics of patients. For example, genetic variations make some people immune to the effects of anticoagulants such as plavix. The presence of certain mutations or DNA sequences in the genome will make it possible to predict the body's reaction to the appropriate drugs with very high accuracy.
And finally, thanks to the possibility of using a CRISPR chip to monitor the binding of the CRISPR protein to certain DNA sequences, it can also be used to test the effectiveness of CRISPR-based DNA editing methods. For example, to verify the correctness of the design of guide DNA sequences.
Article by Reza Hajian et al. Detection of unexplained target genes via CRISPR–Cas9 immobilized on a graphene field-effect transistor is published in the journal Nature Biomedical Engineering.
Evgenia Ryabtseva, portal "Eternal Youth" http://vechnayamolodost.ru based on the materials of the University of California, Berkeley: New CRISPR-powered device detects genetic mutations in minutes.