Functional Study Of VlincRNAs Based On CRISPR/cas13

Long non-coding RNAs(lncRNAs)represent transcripts longer than 200 nucleotides that do not encode proteins.While implicated in virtually all biological processes,the biological significance and the molecular mechanisms of action of these RNAs remain poorly understood and vigorously debated.Reverse-genetics techniques tools have been widely used in attempts to reveal the true biological relevance of lncRNAs.However,a number of subsequent studies have shown that the reverse-genetics techniques used either suffer from non-specific and off target effects and/or can not unambiguously assign the observed phenotypes to the transcripts per se.Moreover,a number of unique features of lncRNA,such as genomic overlap with other functional elements,poorly understood mechanisms of action,alternative processing,nuclear localization,low abundance and so on make the studies challenging.Therefore,new methods and approaches are badly needed to understand the functionality of this promising yet enigmatic class of transcripts.In this project,I aim to study the function of a new class of lncRNAs,the very long intergenic non-coding RNAs(vlincRNAs),using the recently developed CRISPR/Cas13 system which has higher specificity than other methods(such as RNAi)and the ability to act on RNA rather than DNA as the more famous CRISPR/Cas9 system.The latter functionality is critical in unambiguously assigning the phenotypes to the transcripts.We combined this technique with the cell survival challenge caused by anticancer drugs to develop a high-throughput screening assay to study the function of vlincRNAs and potentially any other transcript(coding or non-coding).In this system,we introduced multiple controls to ensure the true phenotype-transcript assignment.Using this system,we found that up to 60% of the tested mRNAsand 64% of vlincRNAs were critical to cancer cell survival in response to anticancer drugs.All in all,this project provides a high-throughput system based on CRISPR / Cas13 that can be used for reverse genetics research of protein coding genes and vlincRNAs.In addition,this project provides direct evidence for biological functional significance of the vlinc class of lncRNAs.