Project Description

1. Project choice

In spring 2020, during the current pandemic, we drew our attention to the hypotheses about possibility of SARS-CoV-2 separation into several genetic variants [1]-[4], the impact of the mutations on the severity of the disease course[5] and the development of drug resistance[6]. We decided to create a detection system that could help health workers and patients to distinct different genotypes of the virus. However, after the first cycle of our Human Practice work, we understood that there were no confirmations of Sars-CoV-2 genotypes significance for its treatment. Therefore, we decided to continue monitoring the emerging articles.

Later, we made a list of common viral diseases, for which the definition of viruses subtypes could have clinical relevance. According to the scientific literature, genotyping could be important for two of the viral diseases that require long-term treatment and that currently have no vaccine, hepatitis C (HCV)[7] and HIV.

After consulting with experts, we chose to work on HCV genotype detection[8]. HСV spreads through contaminated blood and causes liver inflammation, which could lead to cancer. Our literature research showed that a recombinant variant RF2k/1b is common in Russia, unlike Europe and the United States.

2. Detection system choice

In our opinion, one of the most relevant applications of CRISPR/Cas technology are the accurate and efficient nucleic acids detection for clinical diagnosis, genotyping, and determination of RNA-containing viruses, such as SARS-CoV-2, HCV and HIV.

Recently, scientists have learned how to use two Cas proteins, Cas13 and Cas12a, in fast, mobile, and inexpensive diagnostics with high accuracy - in SHERLOCK and DETECTR. Both proteins cut nucleic acids recognized by crRNA, which is complementary to RNA or DNA of a virus. The basic principle implemented in these systems is the utilizing of Cas13 and Cas12a collateral activity right after binding to target sequence (RNA for Cas13, dsDNA for Cas12a)[9],[10]. Reporter oligonucleotides are labeled with a fluorophore at one end and a fluorescence quencher at the other end. These oligonucleotides can be cleaved in presence of target sequence producing fluorescence.

However, Cas12a and Cas13a are large proteins - 1200 amino acids (aa) and 1120, respectively. This causes problems with AAV-mediated delivery of these Cas-proteins’ sequences and complicates their production and purification. On the other hand, a new small Cas protein (980 aa) with collateral activity was recently discovered - CasX (Cas12e). It can help us to overcome described difficulties. Therefore, we chose CasX as a main component of our HCV genotypes detection system.

3. Conclusion

Thus, we develop a fast, portable, and sensitive detection system for HCV genetic variants. In future, our system could be applied to the detection of any other RNA-viruses, whose genotypes influence the treatment or vaccine development. The detection principle is illustrated below.

Project Illustration