The development of molecular biology and biotechnologies made it possible to create a lot of biosensors and diagnostic tests – from fast analyses on COVID-19 to express-tests on pathogens, striking agricultural plants. Scientists from The Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences created a platform DIRECT2, that can give birth to new-generation express tests on the base of system CRISPR-Cas12, and demonstrated its work on the example of DNA Dickeya solani – a bacterium, that harms agricultural plants. The biotechnologists described principles of new platform’s work and its comparison with other methods in the journal Biosensors and Bioelectronics.
Systems from gene section CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), that stores information about previously met viruses, and protein- nuclease of group Cas (CRISPR associated protein) provide the bacteria’s immunity, protecting it from reinfection by the same viruses. Programmability of these systems and marked specificity of recognition of gene target make it possible to use them in the genome editing. In the last years systems CRISPR-Cas began to be used also in biosensors, that recognize certain sequences of DNA and RNA with high sensitivity. Russian scientists created a new platform, combining technology CRISPR-Cas, universal DNA -protein probe and membrane express test, that provides high-fidelity of non-apparatus diagnostics.
Protein Cas12 in complex with guide RNA recognizes DNA-target in the test, comparing its sequence with guide RNA – in the same way as in the bacterial cell there is a process of checking of information about previously met viruses. Then the complex CRISPR- Cas12 cuts the DNA -target and acquires the ability to repeatedly cleave any single-stranded DNA. Using single-stranded DNA with detectable label (DNA -probe) it is possible to register the release of this label, for example, with the help of the membrane test. By this the coloring of the test line appears in the presence of the targeted DNA in the test, and it is absent in the negative tests. The process of testing doesn’t need complex equipment, only minimal operator’s support, that enables to make it in field conditions. However, in the previously made products the undegraded DNA-probe was also connected with test, and it can distort the result of the analysis.
In order to deal with this difficulty, the authors of the study created biosensor, on the carrier of which the probe is placed, that contains DNA-part (single – stranded DNA with functional group – hapten) and protein part (murine antibodies against hapten). Activated be DNA-target protein Cas12 cuts the probe. Released construction is detected by lateral flow test thanks to specific binding of antibody with murine antibodies, placed on the membrane and on the surface of gold nanoparticles. The platform was called DIRECT2 (abbreviated from DNA-Immunoglobulin Reporter Endonuclease Cleavage Test).
Boris Dzatntiev, Professor, Dr. Sci. (Chemistry), Head of Laboratory of Immunobiochemistry of the Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences:
“Modern molecular methods of diagnostics transgressed the point of domination of PCR-testing as the only highly-sensitive instrument. Biotechnology creates new approaches, that significantly shorten testing time and enable to find single viruses or bacteria in the sample. And what’s more important, diagnostics becomes available for carrying directly at bedside, doctor’s office and any other place, that is not an equipped laboratory. Express tests are becoming the reliable alternative to laboratory diagnostics thanks to such new technologies as isothermal amplifications, CRISPR -Cas system of precise recognition of DNA/RNA -targets.
DIRECT2 is the first Russian technology, using highly-sensitive and specific CRISPR-Cas system for diagnostics. Alongside with the publication of our results there was an article of Chinese colleagues, published in the journal Angewandte Chemie International Edition, and also offering the reliable detection of reaction products of CRISPR-Cas12 with the help of testing strip. At present these works can be considered as the beginning of new generation of express-tests with CRISPR-Cas systems.
The platform is a flexible system, it can be changed and developed according to the current task, and adapted to recognition of various pathogens. Moreover, it will be useful to explore other carriers and components of the composite probe.
Irina Safenkova PhD (Biology), senior researcher of Immunobiochemistry of the Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences:
“The user sees the positive result of express test with CRISPR-Cas12 as the appearance of two lines (the test one, T-zone, and the control one, C-zone) on the testing strip. You can see these lines thanks to colored nanoparticles. But the content of the test, molecular processes and the reliability of the obtained result are completely different when using the platform DIRECT2 and tests, used previously.
The ordinary express test with CRISPR-Cas12 works according such scheme: testing strip catches the whole DNA -probe and nanoparticles in C-zone and doesn’t let them into T-zone. DNA- target activates Cas12, that leads to cutting of DNA-probe. Cut DNA – probe becomes the pass for nanoparticles to T-zone. The serious shortcoming of this scheme is the risk of obtaining false-positive result. Nanoparticle can be let into T-zone under the influence of different factors, there is no one-to-one connection between cutting of the probe and coloring of T-zone. In the platform DIRECT2 the appearance and binding of nanoparticles in T-zone, that leads to its coloring, is impossible without cutting of DNA-probe. Such scheme provides high reliability of testing.
The set for carrying DIRECT2 – analysis includes reaction mixture and two components. The first component is composite probe DNA-protein, adsorbed on the carrier in the wells of microplate or on magnetic particles. By cutting the probe, its protein part is released. The second component is a test strip, that detects the protein part of the probe. T-zone is colored by cutting DNA-probe with activated Cas12. C-zone is always colored, independently of the test content.
