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BNAs dramatically improve CRISPR Cas9 specificity

BNAs dramatically improve the specificity of CRISPR-Cas9. 

The addition of BNAs (2′,4′-BNANC[N-Me]) to CRISPR-RNAs (crRNAs) dramatically improves the accuracy in CRISPR-based gene-editing. In this approach, natural crRNA molecules are replaced with synthetic crRNAs containing bridged nucleic acids, or BNAs.

BNA-T (2′,4′-BNANC[N-Me]-T)

Off-target effects or off-target cutting and the generation of additional mutations remain a significant barrier for the use of Cas9-based gene editing. Therefore a number of approaches have been investigated and developed to improve the specificity of Cas9 and to minimize or abolish off-target effects. 

Cromwell et al. in 2018 now report that the incorporation of bridged nucleic acids, BNAs, at specific positions within crRNAs significantly improves Cas9 DNA cleavage specificity in vitro and cells with close to no off-target cleavage. The researcher reasoned that modified crRNAs enhance specificity by impairing the formation of the stable “zipped” conformation during hybridization to off-target sequences.

These results indicate that BNA modified crRNAs improve the specificity of the CRISPR-Cas9 system and illustrate the power of recently developed synthetic nucleic acid technologies to solve problems in enzyme specificity as well.

The research was published in "Nature Communications" in a paper entitled “Incorporation of Bridged Nucleic Acids into CRISPR RNAs Improves Cas9 Endonuclease Specificity.”

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas9 complex is a component of prokaryotic immune systems that is now a widely used tool for genome editing technologies. CRISPR-Cas9 can be used for the generation of gene knock-outs and knock-ins in a variety of organisms, functional genomics studies, epigenetic screens, and proof-of-principle studies that enable the correction of genetic diseases in mammals.

Reference

Cromwell, Christopher R., Sung, Keewon, Park, Jinho, Krysler, Amanda R., Jovel, Juan, Kim, Seong Keun, and Hubbard, Basil P.; 2018. Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity. Nature Communications 9, 1448.


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