RNA-targeting CRISPR-Cas systems have emerged as alternatives to RNA-interference technology to knock down specific RNA transcripts. In particular, Cas13d derived from(CasRx, RfxCas13d) has generated interest due to its superior knockdown efficiencies; however, accumulating evidence indicates that CasRx is prone to inducing transcriptome alterations due to its tendency to cleave bystander RNAs. High-fidelity Cas13d (hfCas13d) derived from CasRx and DjCas13d, an ortholog of Cas13d derived fromsp. UBA7013 (gut metagenome), are two recently identified variants that are superior to CasRx, as they both show a reduced tendency to cleave bystander RNAs. In this study, we created a resource of adeno-associated viral (AAV) vectors designed to deliver Cas13d, including hfCas13d and DjCas13d. We directly compared hfCas13d and DjCas13d for their on- and off-target potential in 293FT and neuro 2A cells. Specifically, we examined their ability to knockdown several endogenous and ectopically expressed transcripts using several different guide RNAs (gRNAs), and we examined knockdown specificity using a combination of reporter assays, RNA integrity analysis, and RNA sequencing (RNA-seq). We report that while both of these enzymes exhibit generally similar levels of knockdown potential, with DjCas13d sometimes outperforming hfCas13d, hfCas13d consistently caused significantly fewer transcriptome alterations when targeting highly expressed genes compared to DjCas13d. Ruminococcus flavefaciens Ruminococcus
