We recently reported two novel tools for precisely controlling and quantifying Cas9 activity: a chemically inducible Cas9 variant (ciCas9) that can be rapidly activated by small molecules and a ddPCR assay for time-resolved measurement of DNA double strand breaks (DSB-ddPCR). Here, we further demonstrate the potential of ciCas9 to function as a tunable rheostat for Cas9 function. We show that a new highly potent and selective small molecule activator paired with a more tightly regulated ciCas9 variant expands the range of accessible Cas9 activity levels. We subsequently demonstrate that ciCas9 activity levels can be dose-dependently tuned with a small molecule activator, facilitating rheostatic time-course experiments. These studies provide the first insight into how Cas9-mediated DSB levels correlate with overall editing efficiency. Thus, we demonstrate that ciCas9 and our DSB-ddPCR assay permit the time-resolved study of Cas9 DSB generation and genome editing kinetics at a wide range of Cas9 activity levels.

中文翻译：

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Cas9介导的DNA双链断裂（DSB）生成和基因组编辑的变阻性控制。

我们最近报道了两种用于精确控制和定量Cas9活性的新颖工具：一种化学诱导的Cas9变体（ciCas9），可以被小分子快速激活，另一种是ddPCR分析法，用于时间分辨的DNA双链断裂（DSB-ddPCR）。在这里，我们进一步证明了ciCas9作为Cas9功能的可变变阻剂的潜力。我们显示，与更严格调控的ciCas9变体配对的新的高效和选择性小分子激活剂扩大了可访问的Cas9活性水平的范围。我们随后证明，可以使用小分子激活剂剂量依赖性地调节ciCas9活性水平，以利于变阻性时程实验。这些研究首次揭示了Cas9介导的DSB水平如何与整体编辑效率相关。因此，