Design of Nucleic Acid Biosensors Based on CRISPR/Cas Systems and Reporter Split Proteins

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.

CRISPR/Cas: A New Tool in the Research of Telomeres and Telomerase as Well as a Novel Form of Cancer Therapy

“…In the presence of an adjacent protospacer motif (PAM) on the opposite strand, the sgRNA is able to direct CRISPR/Cas9 to a target location for cleavage [ 47 ], leading to DNA DSBs ( Figure 2 ) [ 31 , 48 ]. The sequence-specific dsDNA binding and cleavage capabilities of the Cas9/sgRNA or dCas9/sgRNA systems have been used to develop biosensors for nucleic acid assays [ 40 , 47 , 49 ]. Because of characteristics such as adaptability, simplicity, specificity, and effectiveness, CRISPR/Cas9 technology has been extensively employed for genome editing ( Figure 3 ) and has a significant potential for biomedical studies [ 50 , 51 ].…”

Nucleosomes and their complexes in the cryoEM era: Trends and limitations

“…The next stage of nucleosome complexes research might be expanding the range of proteins interacting with nucleosomes to other DNA interacting proteins. For example, there are currently no structural details about the interplay between nucleosome and CRISPR-Cas systems, which are potential candidates for novel therapy approaches and detection systems ( Yarrington et al, 2018 ; Novikov et al, 2021 ).…”