Molecular Virology, Department of Medicine, Imperial College London, London, UK.
Single Molecule Imaging Group, MRC-London Institute of Medical Sciences, London, UK.
Nat Struct Mol Biol. 2019 Mar;26(3):185-192. doi: 10.1038/s41594-019-0188-z. Epub 2019 Feb 25.
CRISPR/Cas9 is a powerful genome-editing tool, but spurious off-target edits present a barrier to therapeutic applications. To understand how CRISPR/Cas9 discriminates between on-targets and off-targets, we have developed a single-molecule assay combining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence-dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule Förster resonance energy transfer (smFRET) and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with ten mismatches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.
CRISPR/Cas9 是一种强大的基因组编辑工具，但非特异性的脱靶编辑是其治疗应用的一个障碍。为了了解 CRISPR/Cas9 如何区分靶标和脱靶，我们开发了一种将光学镊子与荧光相结合的单分子测定法，以监测 λ-DNA 的结合。在低力下，酿脓链球菌 Cas9 复合物特异性地结合并切割 DNA。在更高的力下，许多脱靶结合事件以引导 RNA 序列依赖性的方式反复出现在相同的脱靶位点，这是由 DNA 的机械扭曲驱动的。我们使用单分子Förster 共振能量转移 (smFRET) 和切割测定法表明，即使存在十个错配，DNA 泡也会诱导这些位点的脱靶结合和切割，以及先前鉴定的体内脱靶。我们提出，细胞过程中双链 DNA 的不稳定性（例如转录、复制等）可以使这些隐匿的脱靶位点暴露于 Cas9 活性之下，这凸显了对改进的脱靶预测算法的需求。
