A blood-based miRNA signature with prognostic value for overall survival in advanced stage non-small cell lung cancer treated with immunotherapy

Journal article

Cellular RNAs encode important information regarding cell identity, disease status or exposure to environmental factors. The development of CRISPR-based RNA sensors that are conditionally activated upon detection of endogenous RNA biomarkers would enable restricting CRISPR activity to target cells that were not previously targetable using tissue-specific promoters or targeted delivery approaches. My doctoral work is aiming to develop CRISPR-based RNA sensors by engineering Streptococcus pyogenes Cas9 single-guide RNAs (sgRNAs). sgRNAs are engineered to fold into complex secondary structures that, in the ground state, inhibit their activity. Upon recognising complementary RNA biomarkers, these sgRNAs should become activated, enabling Cas9 to perform its function. In the first part of this project, I implemented a standard Synthetic Biology design-build-test cycle in mammalian HEK293T cells to optimise engineered sgRNA designs. Iterative design optimisations enabled development of a computational pipeline for designing engineered sgRNAs starting from the desired sgRNA and RNA biomarker sequences. Furthermore, insights into the molecular mechanism of RNA detection are also provided. After initial proof-of-concept studies in HEK293T cells, transgenic zebrafish lines were generated to enable rapid screening of different engineered sgRNA designs in vivo. Preliminary results suggest that engineered sgRNAs could be repurposed towards RNA detection in zebrafish embryos. Proposed CRISPR-based sensors design could open up novel avenues towards development of new therapeutic, research and diagnostic applications. However, in order to reach its full potential, further work is still required for optimising this technology in vivo. We still need to explore chemical modifications that stabilise sgRNA designs in vivo, as well as different endogenous RNA classes that can be efficiently detected. Eukaryotic cells express many classes of transcripts of different sizes, expression levels and subcellular localisations, and these factors are expected to significantly impact dynamic ranges of sgRNA activation

Authors: Rajakumar, T; Horos, R; Jehn, J; Schenz, J; Muley, T

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Nature Research
• Journal: npj Precision Oncology
• Volume: 6
• Issue: 1
• Pages: 19-19
• Article number: 19
• Publication date: 2022-03-31
• DOI: 10.1038/s41698-022-00262-y
• EISSN: 2397-768X
• ISSN: 2397-768X
• Language: English
• Keywords: Biomarker; Internal medicine; Cancer; Lung cancer; Oncology; Stage (stratigraphy); Medicine; Biology; Immunotherapy; Cancer immunotherapy