Abstract—

Highly sensitive, specific, rapid, and easy-to-use diagnostic methods for the detection of nucleic acids of pathogens are required for the diagnosis of many human, animal, and plant diseases and environmental monitoring. The approaches based on the use of the natural ability of bacterial CRISPR/Cas9 systems to recognize DNA sequences with a high specificity under isothermal conditions are an alternative to the polymerase chain reaction method, which requires expensive laboratory equipment. The development of the methods for signal registration with the formation of a DNA/RNA/Cas9 protein complex is a separate bioengineering task. In this work, a design was developed and the applicability of a biosensor system based on the binding of two dCas9 proteins with target DNA sequences (without their cutting) and detection of their colocalization using reporter systems based on split enzymes was studied. Using the methods of molecular modeling, possible mutual positions of two dCas9 proteins at a detectable locus of genomic DNA, allowing the split enzyme domains attached to them to interact in an optimal way, were determined. The optimal distances on DNA between binding sites of dCas9 proteins in different orientations were determined, and the dependence of the complex structure on the distance between the binding sites of dCas9 proteins was modeled. Using the methods of bioinformatics, the genomes of a number of viruses (including SARS-CoV-2) were analyzed, and the presence of genomic loci unique to the species, allowing the possibility of landing pairs of dCas9 proteins in optimal positions, was demonstrated. The possibility of a combined use of dCas9 proteins from different bacteria to expand the spectrum of detected loci was analyzed. The results of the work indicate a fundamental possibility of the creation of highly specific nucleic acid biosensors based on a combination of CRISPR/Cas9 technologies and split enzymes.

中文翻译：

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基于CRISPR/Cas系统和报告基因分裂蛋白的核酸生物传感器设计

摘要-

许多人类、动植物病害的诊断和环境监测都需要高灵敏、特异、快速、简便的病原体核酸检测诊断方法。基于使用细菌 CRISPR/Cas9 系统在等温条件下以高特异性识别 DNA 序列的天然能力的方法是聚合酶链反应方法的替代方法，该方法需要昂贵的实验室设备。开发用于形成 DNA/RNA/Cas9 蛋白质复合物的信号配准方法是一项单独的生物工程任务。在这项工作中，开发了一种设计，并研究了基于两个 dCas9 蛋白与目标 DNA 序列（无切割）结合的生物传感器系统的适用性，并研究了使用基于裂解酶的报告系统检测它们的共定位。使用分子建模方法，确定了两个 dCas9 蛋白在基因组 DNA 的可检测位点上可能的相互位置，从而允许连接到它们的分裂酶域以最佳方式相互作用。确定了不同方向的 dCas9 蛋白结合位点之间的 DNA 最佳距离，并模拟了复杂结构对 dCas9 蛋白结合位点之间距离的依赖性。利用生物信息学的方法，分析了多种病毒（包括SARS-CoV-2）的基因组，并且证明了该物种特有的基因组位点的存在，允许将 dCas9 蛋白对定位在最佳位置的可能性。分析了联合使用来自不同细菌的 dCas9 蛋白以扩大检测位点谱的可能性。这项工作的结果表明，基于 CRISPR/Cas9 技术和裂解酶的组合，创建高度特异性核酸生物传感器的基本可能性。