Very fast CRISPR on demand Numerous efforts have been made to improve the temporal resolution of CRISPR-Cas9–mediated DNA cleavage to the hour time scale. Liu et al. developed a Cas9 system that achieved genome-editing manipulation at the second time scale (see the Perspective by Medhi and Jasin). Part of the guide RNA is chemically caged, allowing the Cas9-guide RNA complex to bind at a specific genomic locus without cleavage until activation by light. This fast CRISPR system achieves genome editing at high temporal resolution, enabling the study of early molecular events of DNA repair processes. This system also has high spatial resolution at short time scales, allowing editing of one genomic allele while leaving the other unperturbed. Science, this issue p. 1265; see also p. 1180 Very fast CRISPR on demand enables DNA repair studies at high resolution in space and time at specific genome locations. CRISPR-Cas systems provide versatile tools for programmable genome editing. Here, we developed a caged RNA strategy that allows Cas9 to bind DNA but not cleave until light-induced activation. This approach, referred to as very fast CRISPR (vfCRISPR), creates double-strand breaks (DSBs) at the submicrometer and second scales. Synchronized cleavage improved kinetic analysis of DNA repair, revealing that cells respond to Cas9-induced DSBs within minutes and can retain MRE11 after DNA ligation. Phosphorylation of H2AX after DNA damage propagated more than 100 kilobases per minute, reaching up to 30 megabases. Using single-cell fluorescence imaging, we characterized multiple cycles of 53BP1 repair foci formation and dissolution, with the first cycle taking longer than subsequent cycles and its duration modulated by inhibition of repair. Imaging-guided subcellular Cas9 activation further facilitated genomic manipulation with single-allele resolution. vfCRISPR enables DNA-repair studies at high resolution in space, time, and genomic coordinates.

中文翻译：

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非常快速的按需 CRISPR


按需快速 CRISPR 为了将 CRISPR-Cas9 介导的 DNA 切割的时间分辨率提高到小时时间尺度，人们做出了许多努力。刘等人。开发了一个 Cas9 系统，实现了第二时间尺度的基因组编辑操作（参见 Medhi 和 Jasin 的观点）。部分引导 RNA 被化学笼住，使得 Cas9-引导 RNA 复合物能够结合在特定的基因组位点上，而不会被切割，直到被光激活。这种快速的 CRISPR 系统以高时间分辨率实现基因组编辑，从而能够研究 DNA 修复过程的早期分子事件。该系统在短时间尺度上还具有高空间分辨率，允许编辑一个基因组等位基因，同时保持另一个基因组等位基因不受干扰。科学，本期第 14 页。 1265；另见 p. 1180 极快的按需 CRISPR 能够在特定基因组位置进行高分辨率的空间和时间 DNA 修复研究。 CRISPR-Cas 系统为可编程基因组编辑提供了多功能工具。在这里，我们开发了一种笼状 RNA 策略，允许 Cas9 结合 DNA，但在光诱导激活之前不会裂解。这种方法被称为极快 CRISPR (vfCRISPR)，可在亚微米和秒尺度上产生双链断裂 (DSB)。同步切割改进了 DNA 修复的动力学分析，揭示细胞在几分钟内对 Cas9 诱导的 DSB 做出反应，并且在 DNA 连接后可以保留 MRE11。 DNA 损伤后 H2AX 的磷酸化速度超过每分钟 100 千碱基，最高可达 30 兆碱基。使用单细胞荧光成像，我们对 53BP1 修复灶形成和溶解的多个周期进行了表征，第一个周期比后续周期花费的时间更长，并且其持续时间通过修复抑制进行调节。 成像引导的亚细胞 Cas9 激活进一步促进了单等位基因分辨率的基因组操作。 vfCRISPR 能够在空间、时间和基因组坐标上进行高分辨率的 DNA 修复研究。