Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity.
Christopher R CromwellKeewon SungJinho ParkAmanda R KryslerJuan JovelSeong Keun KimBasil P HubbardPublished in: Nature communications (2018)
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'-BNANC[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 BNANC 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.
Keyphrases
- single molecule
- crispr cas
- genome editing
- atomic force microscopy
- living cells
- dna binding
- circulating tumor
- endothelial cells
- induced apoptosis
- cell cycle arrest
- cell free
- molecular dynamics simulations
- dna methylation
- cell proliferation
- oxidative stress
- induced pluripotent stem cells
- genome wide
- structural basis
- cell death
- high speed
- pluripotent stem cells
- crystal structure