Scientists at the University of Alberta in Canada have developed a technology that can dramatically improve the specificity of CRISPR-Cas9 gene editing. The approach uses synthetic guide molecules known as bridged nucleic acids (BNAs) in place of the system’s native guide RNAs (gRNAs) to direct the Cas9 enzyme to its target DNA sequence, and so reduce off-target DNA cleavage.

“We’ve 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,” claims lead researcher Basil Hubbard, Ph.D., Canada Research Chair in Molecular Therapeutics and assistant professor at the University of Alberta’s Department of Pharmacology. “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.”

The University of Alberta team and colleagues at Seoul National University in Korea, report on the new technology in Nature Communications, in a paper entitled “Incorporation of Bridged Nucleic Acids into CRISPR RNAs Improves Cas9 Endonuclease Specificity.”

The CRISPR-Cas9 system has become a key genome-editing tool that has widespread applications in fields spanning model organisms, functional genomics, and epigenetic screens, and potentially human therapeutics. However, the University of Alberta team points out, the system isn’t foolproof, and while 99% of the time the Cas9 enzyme cuts DNA at precisely the right sequence, about 1% of the time it will cut the DNA at the wrong site. “…the system is not perfectly specific,” Dr. Hubbard comments. “…sometimes it cuts a similar but incorrect gene. However, given that there are trillions of cells in the human body, even one percentage off is quite significant, especially because gene editing is permanent. One wrong cut and a patient could end up with a serious condition like cancer.”

Two noncoding RNA elements are involved in directing the Cas9 enzyme to cleave at its target sequence, the University of Alberta team continues. “The CRISPR-RNA (crRNA) contains a 20-nucleotide (nt) RNA sequence complementary to the target DNA sequence, while the transactivating crRNA (tracrRNA) acts as a bridge between the crRNA and Cas9 enzyme.” Hybridization of these two elements constitutes the gRNA.

The specificity of Cas9 DNA cleavage is highly dependent on the crRNA sequence and target-crRNA folding stability, and mutations within the target sequence can result in cleavage at off-target DNA sequences. However, while various approaches have been used to improve Cas9 specificity, “off-target cutting and generation of accessory mutations remains a significant barrier for Cas9-based gene editing.”

Most of the approaches to improving specificity have involved modifying the Cas9 enzyme itself, but the University of Alberta researchers have instead focused on incorporating synthetic nucleotides—BNAs—at specific locations in crRNAs. “Interestingly, bridged nucleic acids (BNAs) have previously been shown to improve mismatch discrimination in nucleic acid duplexes,” they point out. “We hypothesized that incorporation of these synthetic nucleotides into crRNAs could improve Cas9 DNA cleavage specificity.”

Initial in vitro tests showed that incorporating BNA-modified nucleotides into crRNAs broadly improved Cas9 specificity and reduced off-target cleavage. Encouragingly, the BNA-substituted crRNAs were also compatible with Cas9 variants that scientists have generated independently to try and reduce off-target Cas9 activity. “Strikingly, we found that the combination was additive, resulting in elimination of nearly all off-target activity,” say the researchers commenting on the results from testing one combination of modified crRNA and engineered Cas9 and stating that the BNA-modified crRNAs “can complement the specificity enhancements of next-generation Cas9 variants.” Subsequent tests confirmed that the BNA-modified crRNAs induced lower cleavage rates at off-targets sites in cultured cells, as well as in vitro.

Analyses designed to identify the mechanisms at work indicated that the BNA-substituted crRNAs enhance specificity both by slowing Cas9 kinetics and by impairing formation on off-target sites, of the stable “zipped” conformation that is prerequisite for DNA cleavage. Interestingly, using locked nucleic acids (LNAs) substitutions in the crRNAs instead of BNAs also increased Cas9 specificity, but to a lesser extent than the BNA-substituted crRNAs.

“Overall, these findings unveil a strategy for improving the specificity of the CRISPR-Cas9 system and illustrate the application of recently developed synthetic nucleic acid technologies to solving problems in enzyme specificity,” the researchers conclude. “We anticipate that these findings will directly contribute to the ongoing goal of improving the specificity and safety of genome-editing agents for a wide variety of experimental and clinical applications.…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.”