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

Autor:	Hu W; Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia., Kumar A; Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia.; Diagnostic Genomics, Monash Health Pathology, Monash Medical Centre, Clayton, Victoria, Australia., Ahmed SF; Department of Electrical and Electronic Engineering, The University of Melbourne, Parkville, Victoria, Australia.; Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, Victoria, Australia., Qi S; Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia., Ma DKG; Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia.; Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia., Chen H; Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia., Singh GJ; Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia., Casan JML; Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia., Haber M; Children's Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, Sydney, New South Wales, Australia.; School of Women's and Children's Health, UNSW Sydney, Sydney, New South Wales, Australia., Voskoboinik I; Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia., McKay MR; Department of Electrical and Electronic Engineering, The University of Melbourne, Parkville, Victoria, Australia.; Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, Victoria, Australia., Trapani JA; Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia., Ekert PG; Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia.; Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia.; Children's Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, Sydney, New South Wales, Australia.; School of Women's and Children's Health, UNSW Sydney, Sydney, New South Wales, Australia., Fareh M; Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. mohamed.fareh@petermac.org.; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia. mohamed.fareh@petermac.org.
Jazyk:	angličtina
Zdroj:	Nature structural & molecular biology [Nat Struct Mol Biol] 2024 Nov; Vol. 31 (11), pp. 1702-1716. Date of Electronic Publication: 2024 Jul 01.
DOI:	10.1038/s41594-024-01336-0
Abstrakt:	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. Competing Interests: Competing interests Some findings in this study are subject to a provisional patent deposited by the Peter MacCallum Cancer Center. The authors declare no other competing interests. (© 2024. The Author(s).)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu Full text from SpringerLink