Mar 23, 2020

Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying essential circRNAs

BioRxiv : the Preprint Server for Biology

Abstract

Circular RNAs (circRNAs) are widely expressed, but their functions remain largely unknown. To study circRNAs in a high-throughput manner, short hairpin RNA (shRNA) screens1 have recently been used to deplete circRNAs by targeting their unique back-splicing junction (BSJ) sites. Here, we report frequent discrepancies between shRNA-mediated circRNA knockdown efficiency and the corresponding biological effect, raising pressing concerns about the robustness of shRNA screening for functional circRNAs. To address this issue, we leveraged the CRISPR/Cas13d system2 for circRNAs functional screenings. We optimized a strategy for designing single guide RNAs to deplete circRNAs. We then performed shRNA and CRISPR/Cas13d parallel screenings and demonstrated that shRNA-mediated circRNAs screening yielded a high rate of false positives phenotypes, while optimized CRISPR/Cas13d led to the identification of bona-fide functional circRNAs. Collectively, we developed a specific and reliable approach to functionalize circRNAs in a high-throughput manner.

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Mentioned in this Paper

Genome-Wide Association Study
Malpighian Tubule Principal Cell Differentiation
Size
Nucleotides
Structure
Analysis
Genetic Polymorphism
Single Nucleotide Polymorphism
Silo (Dataset)
Familial Lichen Amyloidosis

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