Application of Detectr for Selective Detection of Bacterial Phytopathogen Dickeya solani Using Recombinant CRISPR-Nuclease Cas12a Obtained by Single-Stage Chromatographic Purification

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The work demonstrates that recombinant CRISPR-nuclease Cas12a, purified after heterologous expression with a simplified method using single-stage metal-chelate chromatography, can be successfully utilized in DETECTR technology. The combination of CRISPR-nuclease Cas12a obtained by such way with recombinase polymerase amplification (RPA) allowed one to ensure the selectivity of detection of Dickeya solani — the dangerous bacterial phytopathogen causing the potato disease known as “blackleg” — against closely related and unrelated bacterial phytopathogens with a limit of detection of 1 copy of the bacterial genome per amplification reaction. The result can be determined visually, without the use of complex instrumental methods, by changing the color of the reaction sample when illuminated with blue light that creates the basis for development of field DNA diagnostics of D. solani. The use of simplified chromatographic purification will significantly reduce the time and resources required to obtain a functionally active CRISPR-nuclease Cas12a for development and production of DNA diagnostics based on DETECTR technology.

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作者简介

L. Kurbatov

N.V. Orekhovich Institute of Biomedical Chemistry

编辑信件的主要联系方式.
Email: kurbatovl@mail.ru
俄罗斯联邦, Moscow, 119121

S. Radko

N.V. Orekhovich Institute of Biomedical Chemistry; University of Tyumen, West Siberia Interregional Research and Educational Center

Email: radkos@yandex.ru
俄罗斯联邦, Moscow, 119121; Tyumen, 625003

S. Khmeleva

N.V. Orekhovich Institute of Biomedical Chemistry

Email: kurbatovl@mail.ru
俄罗斯联邦, Moscow, 119121

K. Ptitsyn

N.V. Orekhovich Institute of Biomedical Chemistry

Email: kurbatovl@mail.ru
俄罗斯联邦, Moscow, 119121

O. Timoshenko

N.V. Orekhovich Institute of Biomedical Chemistry

Email: kurbatovl@mail.ru
俄罗斯联邦, Moscow, 119121

A. Lisitsa

N.V. Orekhovich Institute of Biomedical Chemistry; University of Tyumen, West Siberia Interregional Research and Educational Center

Email: kurbatovl@mail.ru
俄罗斯联邦, Moscow, 119121; Tyumen, 625003

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2. Fig. 1. PAGE of the homogenate of E. coli cells expressing the recombinant CRISPR nuclease Cas12a and the eluate fractions obtained during purification of the target protein by metal chelate chromatography on Protino Ni-TED1000 columns: M — molecular weight markers, 1 — cell homogenate, 2–6 — eluate fractions, 7 — pooled fractions after dialysis. The target protein is shown by arrow I, the main contaminant protein is shown by arrow II.

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3. Fig. 2. Results of electrophoretic analysis of RPA products for phytopathogen strains (Table 2) (a) and testing of RPA products with the gRNA/Cas12a complex (b): M — DNA standards, 1 — P. carotovorum ssp. carotovorum, 2 — P. odoriferum, 3 — C. michiganensis ssp. sepedonicus, 4 — P. brasiliense (strain 466), 5 — P. brasiliense (strain 497), 6 — D. solani. Genomic DNA 0.5 pg per RPA sample. The target amplification product is indicated by the arrow. b — Characteristic dependencies of the difference in fluorescence of the sample with the addition of RPA products and the control sample without the addition of RPA products (F–F0, c.u.) on the incubation time (min). (1 µl of RPA sample per test sample with gRNA-3/Cas12a. Concentration of gRNA-3 is 30 nM, concentration of Cas12a is 30 nM.)

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4. Fig. 3. Results of electrophoretic analysis of RPA products for different amounts of D. solani bacterial genomes in the presence of 1 ng of potato DNA (a) and testing of RPA products with the gRNA-3/Cas12a complex (b): M — DNA standards, 1–5 — number of copies of bacterial genomes per RPA sample 1, 10, 100, 1000, and 10000, respectively. The target amplification product is indicated by an arrow. b — Characteristic dependencies of the difference in fluorescence of the sample with the addition of RPA products and the control sample without the addition of RPA products (F–F0, conventional units) on the incubation time (min). (1 μl of RPA sample per test tube with gRNA/Cas12a. Concentration of gRNA-3 – 30 nM, concentration of Cas12a – 30 nM).

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5. Fig. 4. Characteristic dependencies of the difference in fluorescence of the sample with the addition of RPA products and the control sample without the addition of RPA products (F–F0, c.u.) on the incubation time (min): 1 and 2 — concentrations of CRISPR nuclease Cas12a of 90 and 150 nM, respectively. Molar ratio of gRNA-3/Cas12a = 1. (1 μl of RPA sample per test sample with gRNA-3/Cas12a. 1 copy of the bacterial genome per RPA sample). In the inset: 1 — control sample, 2 — sample as in curve 2 in Fig. 4. Irradiation with blue light (400–500 nm), orange filter.

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