New-Generation Tests. Russian Scientists Created a Platform for New-Generation Express-Tests on the Base of Crispr-cas12

Newswise – The development of molecular biology and biotechnologies has enabled the development of many biosensors and diagnostic tests – from rapid analysis of COVID-19 to express tests for pathogens and conspicuous agricultural plants. Scientists of the Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences have created a platform DIRECT2, which can create express tests of a new generation based on the CRISPR-Cas12 system, and demonstrated their work using DNA as an example Dickeya solani – a bacterium that damages crops. The biotechnologists described the principles of the new platform’s work and how it compares to other methods in the journal Biosensors and Bioelectronics.

Systems from the gene section CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which stores information about previously hit viruses, and protein nuclease of the Cas group (CRISPR-associated protein) ensure the immunity of the bacterium and protect it from re-infection by the same viruses. The programmability of these systems and the high specificity of gene target recognition make their use in genome editing possible. In recent years, CRISPR-Cas systems have also been used in biosensors that detect specific DNA and RNA sequences with high sensitivity. Russian scientists have created a new platform combining CRISPR-Cas technology, the universal DNA-protein probe and the Membrane Express test, which enables high-precision diagnostics without any equipment.

The protein Cas12 in complex with the guide RNA in the test recognizes the DNA target by comparing its sequence with the guide RNA – just like in the bacterial cell there is a process of checking the information about previously hit viruses. Then the CRISPR-Cas12 complex cuts the DNA target and acquires the ability to repeatedly cleave any single-stranded DNA. In the case of single-stranded DNA with a detectable label (DNA probe), it is possible to register the release of this label using the membrane test, for example. As a result, the color of the test line appears in the presence of the target DNA in the test and is absent in the negative tests. The testing process requires no complex equipment but minimal operator assistance, allowing it to be performed under field conditions. However, in the products manufactured to date, the undegraded DNA probe was also connected to the test and can falsify the result of the analysis.

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To deal with this difficulty, the authors of the study have developed a biosensor on the support of which the probe is placed, which contains a DNA part (single-stranded DNA with a functional group – hapten) and a protein part (murine antibodies to hapten ). The activated DNA target protein Cas12 cuts the probe. The released construction is detected by the lateral flow test thanks to the specific binding of antibodies with murine antibodies placed on the membrane and on the surface of gold nanoparticles. The platform was called DIRECT2 (short for DNA Immunoglobulin Reporter Endonuclease Cleavage Test).

Boris Dzatntiev, Professor, Dr. science (Chemistry), Head of the Laboratory of Immunobiochemistry of the Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences:

“Modern molecular biological methods of diagnostics have exceeded the dominance of PCR testing as the only highly sensitive instrument. Biotechnology creates new approaches that significantly reduce testing time and make it possible to find individual viruses or bacteria in the sample. And more importantly, diagnostics become available to carry right at the bedside, in the doctor’s office, and anywhere else that isn’t an equipped laboratory. Express tests become a reliable alternative to laboratory diagnostics thanks to new technologies such as isothermal amplification, CRISPR-Cas system for precise detection of DNA/RNA targets.

DIRECT2 is the first Russian technology to use a highly sensitive and specific CRISPR-Cas system for diagnosis. Parallel to the publication of our results, an article by Chinese colleagues appeared in the journal Angewandte Chemie International Edition, which also offers the reliable detection of CRISPR-Cas12 reaction products using test strips. Currently, this work can be seen as the beginning of a new generation of express tests using CRISPR-Cas systems.

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The platform is a flexible system that can be changed and further developed depending on the current task and adapted to the detection of different pathogens. In addition, it will be useful to study other supports and components of the assembled probe.

Irina Safenkova PhD (Biology), Senior Researcher in Immunobiochemistry of the Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences:

“The user sees the positive result of the express test with CRISPR-Cas12 as the appearance of two lines (the test line, T-zone, and the control line, C-zone) on the test strip. You can see these lines thanks to colored nanoparticles. However, the content of the test, the molecular processes and the reliability of the result obtained are completely different when using the DIRECT2 platform and the tests used previously.

The usual rapid test with CRISPR-Cas12 works according to this scheme: test strip catches all DNA probe and nanoparticles in the C-zone and does not let them into the T-zone. DNA target activates Cas12, resulting in cutting of the DNA probe. Cut DNA – probe becomes passage for nanoparticles to T-zone. The serious shortcoming of this scheme is the risk of getting a false positive result. Nanoparticles can be let into the T-zone under the influence of various factors, there is no one-to-one connection between cutting the probe and staining the T-zone. In the DIRECT2 platform, the appearance and binding of nanoparticles in the T-zone, leading to their staining, is impossible without cutting the DNA probe. Such a scheme provides high reliability of testing.

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The kit for carrying the DIRECT2 analysis includes the reaction mixture and two components. The first component is an assembled probe DNA protein adsorbed onto the support in the wells of the microplate or onto magnetic particles. Cutting the probe releases its protein portion. The second component is a test strip that detects the protein content of the probe. The T-zone is stained by cutting a DNA probe with activated Cas12. C-Zone is always colored, regardless of test content.

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