Development of CRISPR-based biosensors for detection of disease-related genes

Abstract

CRISPR complexes are a type of adaptative immune system found in bacteria, working mainly by targeting the cleavage of invasive sequences of DNA. The simplified mechanism by which the CRISPR/Cas system operates consists of the process that, after the invading DNA enters the cell for the first time, it is degraded and incorporated into specific locations of the host genetic material in the form of spacers, from the cleavage of its nucleic acid sequence into small pieces. Once incorporated into the bacterial genome, these spacers are transcribed, subsequently processing into several smaller RNAs, each with a spacer, called crRNA (CRISPR-derived RNA). Subsequently, the crRNA forms an interference complex with the Cas9 protein that can recognize and destroy the exogenous sequence in the event of a second invasion, ending the infection before it even starts.Understanding how this mechanism works led to great scientific and technological knowledge, arising from the possibility of directing the endonuclease by its guide RNA, for the cleavage of specific targets, making it an important tool also for the diseases and biomarkers diagnosis. Although, some interesting platforms have been developed with Cas9, the discovery of Cas13a and Cas12a proteins made a revolution in the field of nucleic acid detection. These enzymes, Cas12a and Cas13a, have additional trans (collateral) cleavage DNase/RNase activity that acts on non-target ss/sdDNA and ssRNA sequences, respectively, activated by the presence of the target sequences. The collateral cleavage property of these enzymes is the basic principle for diagnostic tools.The combination of this endonuclease cleavage mechanism with other amplification methods allowed the development of techniques for disease detection, in which the CRISPR/Cas systems act as programmable and integrated biomolecular components that achieve accurate recognition, in addition to high sensitivity for biosensing. Therefore, using the CRISPR technology, based on Cas12a and Cas13a endonucleases, the present project aims the development of a universal platform for application in highly sensitive and selective electrochemical biosensors for the simultaneous determination of genetic material of viruses and bacteria with a simple approach for the fabrication of point-of-care devices.First, nucleic acids, biomarkers, receptors, and electrochemical probes will be properly designed and manufactured. Oligonucleotides compatible with the desirable biomarker will be selected and immobilized at the sensor platform, obtaining a CRISPR/Cas-based biosensor. All steps will be optimized and properly characterized. Firstly, the detection of the bacterial target DNA biomarkers using the Cas12a enzyme will be performed. In the first stage, single marker biosensors will be developed. After establishing the biomarkers, the proposed analysis method will be studied in the presence of genetic material in a buffer solution, artificial biological sample, or human blood serum. In addition, the sensor validation will be performed using ELISA and PCR techniques. Following the primary construction of the biosensor, studies will be performed to modify the electrode surface with metallic nanoparticles and carbon-based materials (graphene, graphene oxide, MWCNT), to evaluate the increase in sensitivity of the biosensor and to immobilize ssDNA sequences and ssRNA at the sensor surface. Finally, with the sensor platform already developed and validated, tests on printed devices will be performed to build a simple and inexpensive point-of-care system for detecting genetic material. (AU)