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

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 (

SUBMITTER: Hu W 

PROVIDER: S-EPMC11564092 | biostudies-literature | 2024 Nov

REPOSITORIES: biostudies-literature