Temperature Dependence of Collateral Activity of Thermostable Recombinant CRISPR Nucleases Cas12b Obtained by One-Step Purification with Metal-Chelate Chromatography after Heterologous Expression

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Published: Oct 1, 2025
DOI: https://doi.org/10.18097/BMCRM00284
Keywords:
recombinant CRISPR nuclease Cas12b; single-stage purification; collateral nuclease activity; temperature dependence

Main Article Content

L.K. Kurbatov
Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119435 Russia
O.S. Timoshenko
Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119435 Russia
S.A. Khmeleva
Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119435 Russia
K.G. Ptitsyn
Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119435 Russia
E.V. Suprun
Chemistry Faculty of M.V. Lomonosov Moscow State University, Lenin Hills, 1/3, Moscow, 119991 Russia
S.P. Radko
Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119435 Russia
A.V. Lisitsa
Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119435 Russia

Abstract

Thermostable CRISPR/Cas nucleases are considered as promising enzymes for development of next-generation DNA diagnostics by coupling loop-mediated isothermal amplification of nucleic acids (LAMP) with selective CRISPR/Cas detection of specific amplicons. In this paper, we present the results of testing the collateral activity of CRISPR nuclease AapCas12b and three variants of CRISPR nuclease BrCas12b (wild type and two mutants) obtained using simplified purification in the typical temperature range of LAMP - from 56°C to 72°C. It was shown that the use of one-step metal-chelate chromatography by excluding a stage of enzymatic removal of N-terminal sequences translated together with the target protein allows for obtaining recombinant CRISPR nucleases BrCas12b with a sufficiently high level of collateral activity. Temperature dependences of collateral activity differed among the studied BrCas12b variants. The obtained results can be useful in selecting thermostable CRISPR nucleases Cas12b for development of test systems based on a combination of LAMP and CRISPR/Cas detection.

Article Details

How to Cite
Kurbatov, L., Timoshenko, O., Khmeleva, S., Ptitsyn, K., Suprun, E., Radko, S., & Lisitsa, A. (2025). Temperature Dependence of Collateral Activity of Thermostable Recombinant CRISPR Nucleases Cas12b Obtained by One-Step Purification with Metal-Chelate Chromatography after Heterologous Expression. Biomedical Chemistry: Research and Methods, 8(3), e00284. https://doi.org/10.18097/BMCRM00284
Issue
Vol. 8 No. 3 (2025)
Section
EXPERIMENTAL RESEARCH

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