Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 29654299.
- Also identified by DOI 10.1038/s41467-018-03927-0 and PMC identifier 5899152.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
Off-target DNA cleavage is a paramount concern when applying CRISPR-Cas9 gene-editing technology to functional genetics and human therapeutic applications. Here, we show that incorporation of next-generation bridged nucleic acids (2',4'-BNA<sup>NC</sup>[N-Me]) as well as locked nucleic acids (LNA) at specific locations in CRISPR-RNAs (crRNAs) broadly reduces off-target DNA cleavage by Cas9 in vitro and in cells by several orders of magnitude. Using single-molecule FRET experiments we show that BNA<sup>NC</sup> incorporation slows Cas9 kinetics and improves specificity by inducing a highly dynamic crRNA-DNA duplex for off-target sequences, which shortens dwell time in the cleavage-competent, "zipped" conformation. In addition to describing a robust technique for improving the precision of CRISPR/Cas9-based gene editing, this study illuminates an application of synthetic nucleic acids.
Medical subject headings
- CRISPR-Cas Systems
- Clustered Regularly Interspaced Short Palindromic Repeats
- DNA
- RNA
- RNA, Guide, CRISPR-Cas Systems