The development of precise RNA editing tools is essential for the advancement of RNA therapeutics. CRISPR-PspCas13b (PspCas13b) is a programmable RNA nuclease predicted to offer a superior targeting specificity thanks to its 30-nucleotide spacer sequence. However, PspCas13b design principles and its on-target, off-target, and collateral activities remain poorly characterized. Here, we present Single-Base Tiled screen (SiBTil) using over 200 crRNAs and computational analyses, which identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. In-vitro biochemical cleavage assays and target RNA-protein profiling in human cells show that de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient crRNAs. The potent RNA silencing activity of PspCas13b obtained with optimised design is solely dependent on its nuclease domains, as catalytically inactive enzyme (dPspCas13b) or crRNA alone failed to show any silencing activity. These validated design principles, which we integrated into an online tool (https://cas13b.github.io/), can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer-target mutagenesis we conducted in this study revealed that PspCas13b can tolerate only up to four consecutive or non-consecutive mismatches and thus requires ~26-nucleotide basepairing with the target to activate its nuclease domains and trigger targeted RNA degradation, highlighting its superior specificity compared to other RNA or DNA interference tools. Based on this targeting resolution, we predicted an extremely low probability of PspCas13b off-targeting other cellular RNA transcriptome-wide. Indeed, proteomic and transcriptomic analysis validated this prediction and showed that, unlike other Cas13 orthologues, PspCas13b exhibits potent on-target activity and lacks any off-targeting or collateral effects in human 293 HEK cells. In summary, this study provides novel design principles for PspCas13b that enable highly efficient and collateral-free RNA degradation in human cells.