Progettazione di un sistema CRISPR/Cas13 per identificare lncRNA funzionali nel cancro

LncRNAs are a class of ribonucleic acids longer than 200 nucleotides that are still poorly characterized. Their dysregulation has been associated with several diseases, including several types of cancer. This makes them of potential importance in cancer. For this reason, functional genomics studies can be of great help in characterizing the function of this class of molecules. Among the systems used to perform knock-down experiments, CRISPR/Cas13 is very promising, because of its ability to suppress the expression of specific RNAs based on sequence complementarity. Therefore, we have developed a CRISPR/Cas13 system for use in high throughput screening (HTS). We generated 60 cancer cell lines expressing CasRx (Ruminococcus flavefaciens Cas13) in a stable manner with high silencing efficiency. We merged transcripts from all available lncRNA databases, both manually curated and computationally constructed, and also added information on evolutionary conservation based on 6 articles that have studied evolutionary conservation of lncRNAs. Given the similarity of groups of transcripts arising from the same gene locus, likely originating from the same initial transcript by RNA processing, we created lncRNA families that group similar RNAs together into a single family, which will then be used to design guide RNAs. We then created a program that, starting from a scoring software for Cas13 sgRNAs written by another laboratory, allows sgRNAs to be designed from an input in genomic regions or sequences, giving the possibility to choose different parameters on which to base to guide design. Among the nearly 100,000 lncRNAs in our list, we selected 24172 based on expression level. We mapped RNA-seq from solid tumor-derived cell lines from CCLE (Cancer Cell Line Encyclopedia) and selected a group of lncRNAs most expressed on average in all cell lines, a group expressed in specific tissues, and a group in specific cell lines, and we also selected some of the most expressed conserved transcripts. We then used the program described above to design a sgRNA library with the highest possible score and following a diverse set of parameters, also performing a step of removing off-targets. To validate our sgRNA library we performed an HTS experiment to observe the behavior of sgRNAs and controls. The system we created showed to be promising for future study of lncRNA functionality in cancer. In particular, the use of CRISPR/Cas13 allows us to limit the off-target effect and easily target lncRNAs acting in the nucleus. Given the characteristic cell type-specific expression of many lncRNAs, the generation of 60 stably expressing CRISPR/Cas13 cancer cell lines to perform HTS experiments is of great utility for a large study that aims to elucidate more clearly how lncRNAs function in tumors, and the use of transcripts from multiple databases and the maintenance of storage information allow us to minimize the bias due to the large difference and low concordance we find in lncRNA databases. Our sgRNA library is the largest currently available, as the previous largest library for lncRNA targeted 16401 transcripts. In addition, our sgRNA library includes 54 of the 76 lncRNAs so far thought to be functional in cancer, and those that are not present cannot be targeted because of their characteristics. Finally, the HTS experiment performed in this study suggests that our sgRNA library is well designed, although there is still room for improvement.