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

Abstract

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.