Department of Pharmacology, University of Alberta, Edmonton, AB, T6G 2R7, Canada.
Department of Chemistry, Seoul National University, Seoul, 08826, Republic of Korea.
Nat Commun. 2018 Apr 13;9(1):1448. doi: 10.1038/s41467-018-03927-0.
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'-BNA[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 BNA 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.
在将 CRISPR-Cas9 基因编辑技术应用于功能遗传学和人类治疗应用时，脱靶 DNA 切割是一个首要关注的问题。在这里，我们表明在 CRISPR-RNAs（crRNAs）中的特定位置掺入下一代桥接核酸（2'，4'-BNA[N-Me]）和锁核酸（LNA）可大大降低 Cas9 在体外和细胞中的脱靶 DNA 切割。使用单分子 FRET 实验，我们表明 BNA 掺入通过诱导针对脱靶序列的高度动态 crRNA-DNA 双链体来减缓 Cas9 动力学并提高特异性，从而缩短在切割有效的“zipped”构象中的停留时间。除了描述一种提高基于 CRISPR/Cas9 的基因编辑精度的稳健技术外，这项研究还阐明了合成核酸的一种应用。
