Chemically synthesized guide RNAs can direct CRISPR-CasRx cleavage of circRNAs with high efficiency and specificity

In this study, we demonstrate that CRISPR/RfxCas13d (CasRx)-mediated circRNA depletion is, for the circRNAs studied, more efficient than Argonaute 2-dependent short hairpin RNA (agoshRNA)-mediated depletion and with fewer off-target effects on the linear host RNAs. We also designed and optimally used synthetic guide RNAs (syn-gRNAs) in combination with CasRx to efficienctly deplete circRNA ciRS-7. None of the knockdown strategies tested (pre-gRNA, gRNA, syn-gRNA and agoshRNA) showed any significant off-target effects at the transcriptomics level. Differential expression analysis was performed for each KD setup versus non-targeting control (three biological replicates of each). In conclusion, CasRx-mediated circRNA knockdown using both vector-based and syn-gRNA strategies is a useful tool for future studies on circRNA functions. Overall design: Off-targeting assay of four different KD approaches (pre-gRNA, gRNA, syn-gRNA, and agoshRNA) upon ciRS-7 depletion in SH-SY5Y cells used here could lead to more general off-target effects. We subjected samples from each ciRS-7 KD and the corresponding NTC controls (pre-gRNA, gRNA, syn-gRNA, and agoshRNA) to mRNA sequencing, generating 30 to 35 million reads per sample. Differential expression analysis was performed for each KD setup versus NTC using DESeq2.