Artificial RNAs Increase Precision of Gene-Editing System

Incorporation of next-generation bridged nucleic acids at specific locations in CRISPR-RNAs (crRNAs) broadly reduces off-target DNA cleavage by Cas9 - a major drawback slowing the development of genome editing clinical applications - in vitro and in cells by several orders of magnitude.

CRISPR/Cas9 is regarded as the cutting edge of molecular biology technology. CRISPRs (clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. Since 2013, the CRISPR/Cas9 system has been used in research for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation. By delivering the Cas9 enzyme and appropriate guide RNAs (sgRNAs) into a cell, the organism's genome can be cut at any desired location. The conventional CRISPR/Cas9 system is composed of two parts: the Cas9 enzyme, which cleaves the DNA molecule and specific RNA guides that shepherd the Cas9 protein to the target gene on a DNA strand.

Bridged nucleic acids (BNAs) are modified RNA nucleotides. They are sometimes also referred to as constrained or inaccessible RNA molecules. BNA monomers can contain a five-membered, six-membered, or even a seven-membered bridged structure with a “fixed” C3’-endo sugar puckering. The bridge is synthetically incorporated at the 2’, 4’-position of the ribose to afford a 2’, 4’-BNA monomer. The monomers can be incorporated into oligonucleotide polymeric structures using standard phosphoamidite chemistry. BNAs are structurally rigid oligo-nucleotides with increased binding affinities and stability.

Off-target DNA cleavage is a paramount concern when applying CRISPR-Cas9 gene-editing technology to functional genetics and human therapeutic applications. In an effort to reduce the degree of off-target cleavage generated by CRISPR-Cas9, investigators at the University of Alberta (Edmonton, Canada) reported in the April 13, 2018, online edition of the journal Nature Communications that they had used single-molecule fluorescence resonance energy transfer (FRET) experiments to show that BNANC incorporation slowed Cas9 kinetics and improved specificity. These effects were achieved by inducing a highly dynamic crRNA-DNA duplex for off-target sequences, which shortened dwell time in the cleavage-competent, “zipped” conformation.

"We have discovered a way to greatly improve the accuracy of gene-editing technology by replacing the natural guide molecule it uses with a synthetic one called a bridged nucleic acid, or BNA," said senior author Dr. Basil Hubbard, assistant professor of pharmacology at the University of Alberta. "What researchers have realized is that this system can be programmed to cut a specific DNA sequence in a human cell also, allowing us to edit our genes. One of the main issues, however, is that the system is not perfectly specific--sometimes it cuts a similar but incorrect gene. Our research shows that the use of bridged nucleic acids to guide Cas9 can improve its specificity by over 10,000 times in certain instances--a dramatic improvement."

Related Links:
University of Alberta