Single-base tiled screen unveils design principles of PspCas13b for potent and off-target-free RNA silencing

Hu, W; Kumar, A; Ahmed, SF; Qi, S; Ma, DKG; Chen, H; Singh, GJ; Casan, JML; Haber, M; Voskoboinik, I; McKay, MR; Trapani, JA; Ekert, PG; Fareh, M

Author(s): Hu, W; Kumar, A; Ahmed, SF; Qi, S; Ma, DKG; Chen, H; Singh, GJ; Casan, JML; Haber, M; Voskoboinik, I; McKay, MR; Trapani, JA; Ekert, PG; Fareh, M;
Details: Publication Year 2024-11,Volume 31,Issue #11,Page 1702-1716
Journal Title: Nature Structural & Molecular Biology
Publication Type: Research article
Abstract: The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized. Here, we present single-base tiled screening and computational analyses that identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. We show that the de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient CRISPR RNAs (crRNAs). These validated design principles (integrated into an online tool, https://cas13target.azurewebsites.net/ ) can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer-target mutagenesis revealed that PspCas13b can tolerate only up to four mismatches and requires ~26-nucleotide base pairing with the target to activate its nuclease domains, highlighting its superior specificity compared to other RNA or DNA interference tools. On the basis of this targeting resolution, we predict an extremely low probability of PspCas13b having off-target effects on other cellular transcripts. Proteomic analysis validated this prediction and showed that, unlike other Cas13 orthologs, PspCas13b exhibits potent on-target activity and lacks collateral effects.
Publisher: Springer Nature
Keywords: Humans; *CRISPR-Cas Systems; RNA Interference; HEK293 Cells; Clustered Regularly Interspaced Short Palindromic Repeats; RNA Editing; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Department(s): Laboratory Research
Publisher's Version: https://doi.org/10.1038/s41594-024-01336-0
Open Access at Publisher's Site: https://doi.org/10.1038/s41594-024-01336-0
Terms of Use/Rights Notice: Refer to copyright notice on published article.

Creation Date: 2024-12-12 03:29:04

Last Modified: 2024-12-12 03:29:12