Department of Chemistry, University of Washington , Seattle, Washington 98195, United States.
Department of Genome Sciences, University of Washington , Seattle, Washington 98195, United States.
ACS Chem Biol. 2018 Feb 16;13(2):438-442. doi: 10.1021/acschembio.7b00652. Epub 2017 Sep 15.
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.
我们最近报道了两种用于精确控制和量化 Cas9 活性的新工具：一种可被小分子快速激活的化学诱导型 Cas9 变体（ciCas9），以及一种用于实时测量 DNA 双链断裂（DSB-ddPCR）的 ddPCR 测定法。在这里，我们进一步证明了 ciCas9 作为 Cas9 功能的可调电阻器的潜力。我们表明，一种新型高效且选择性的小分子激活剂与调控更严格的 ciCas9 变体配对，扩展了可访问的 Cas9 活性水平范围。随后，我们证明 ciCas9 活性水平可以通过小分子激活剂进行剂量依赖性调节，从而促进变阻器时程实验。这些研究首次深入了解 Cas9 介导的 DSB 水平与整体编辑效率的相关性。因此，我们证明 ciCas9 和我们的 DSB-ddPCR 测定法允许在广泛的 Cas9 活性水平下实时研究 Cas9 DSB 的产生和基因组编辑动力学。
