Post-transcriptional RNA processing is a fundamental biological process that is important for regulation of most aspects of life. Circular RNA (circRNA), produced from precursor mRNA back-splicing, was shown to be abundant across diverse cell types and play vital roles in cancer progression. However, the absence of tools to systematically perturb circRNAs has hindered the study of circRNA function. The primary objective of this study is to develop an approach that enables specific knockdown of circRNA using both RNA-targeting CRISPR-Cas13 and short hairpin RNA (shRNA) to systematically identify circRNAs that are required for cell proliferation using high-throughput screening.
Initially we designed gRNAs/shRNAs to target different sites of the back-spliced junction of abundant circRNAs and measured the circRNA knockdown specificity and efficiency using both inward and outward primers in HEK293T cells. After confirming the specific circRNA knockdown, gRNAs/shRNAs were packaged into lentiviruses, which were used to infect cancer cells selected for subsequent functional screening.
Based on the rules for optimal targeting of circRNA, we designed and generated genome-wide pooled gRNA/shRNA libraries. Following the delivery of the lentiviral pooled gRNA/shRNA library, cells were passaged for 21 days, after which the gRNA/shRNA abundance of each cell line was measured using Next Generation Sequencing. MAGeCK algorithm was adopted to measure the knockdown performance of each gRNA/shRNA. Our preliminary data has identified novel essential circRNAs, which have independent functions to their expressed genes. More cell lines of colon, pancreatic, skin and brain cancers will be screened to identify circRNAs that are tissue-specific, mutation-specific and signalling pathway specific.
CircRNAs are poorly characterised non-coding RNAs which play important roles in the development of human diseases. This project will provide not only a systematic understanding of the cellular processes associated with circRNAs but also a powerful framework for studies utilising circRNAs as biomarkers in disease diagnosis and treatment.