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′-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.
Highlights
Off-target DNA cleavage is a paramount concern when applying Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas[9] gene-editing technology to functional genetics and human therapeutic applications
Previous work has demonstrated that local mismatch discrimination can be improved in DNA–DNA hybrids when locked nucleic acids (LNA) are incorporated in the vicinity of mismatched bases, with an LNA triplet centered on the mismatch yielding the best results[24]
Our results indicate that incorporation of BNANC or LNA nucleotides within the central region of crRNAs substantially increases specificity in the protospacer adjacent motif (PAM)-proximal and PAM-distal regions (Figs. 1, 2 and Supplementary Figs. 1–4, 16–18)
Summary
Off-target DNA cleavage is a paramount concern when applying CRISPR-Cas[9] gene-editing technology to functional genetics and human therapeutic applications. 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 Cas[9] in vitro and in cells by several orders of magnitude. Several groups have engineered highly specific variants of Cas[9], such as eSpCas[919], SpCas9-HF120, and HypaCas[921], by mutating residues on Cas[9] involved in the formation of non-specific DNA interactions Despite these advances, off-target cutting and generation of accessory mutations remains a significant barrier for Cas9-based gene editing[16]. LNAs display improved base stacking and thermal stability compared to RNA, resulting in highly efficient binding to complementary nucleic acids and improved mismatch discrimination[24,29] They display enhanced nuclease resistance[29]. We show that incorporation of BNANCs and LNAs at specific positions within crRNAs broadly improves Cas[9] DNA cleavage specificity
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