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Abstract：
CRISPR/Cas9 is widely used for precise mutagenesis through targeted DNA double-strand breaks (DSBs) induction followed by error-prone repair. A better understanding of this process requires measuring the rates of cutting, error-prone, and precise repair, which have remained elusive so far. Here, we present a molecular and computational toolkit for multiplexed quantification of DSB intermediates and repair products by single-molecule sequencing. Using this approach, we characterize the dynamics of DSB induction, processing and repair at endogenous loci along a 72 h time-course in tomato protoplasts. Combining this data with kinetic modeling reveals that indel accumulation is determined by the combined effect of the rates of DSB induction processing of broken ends, and precise versus error repair. In this study, 64-88% of the molecules were cleaved in the three targets analyzed, while indels ranged between 15-41%. Precise repair accounts for most of the gap between cleavage and error repair, representing up to 70% of all repair events. Altogether, this system exposes flux in the DSB repair process, decoupling induction and repair dynamics, and suggesting an essential role of high-fidelity repair in limiting the efficiency of CRISPR-mediated mutagenesis.
摘要：
CRISPR/Cas9广泛用于通过靶向DNA双链断裂(DSB)诱导随后的易错修复进行精确诱变。更好地理解这个过程需要测量切割的速度，容易出错，和精确的修复，到目前为止仍然难以捉摸。这里,我们提出了一种分子和计算工具包,用于通过单分子测序对DSB中间体和修复产物进行多重定量.使用这种方法，我们描述了DSB诱导的动力学，在番茄原生质体中沿72小时的时间过程在内源位点进行加工和修复。将这些数据与动力学模型相结合，表明indel积累是由DSB诱导加工断端速率的综合作用决定的，和精确与错误修复。在这项研究中,在分析的三个靶标中，有64-88％的分子被裂解，而indel介于15-41%之间。精确修复占据了分裂和错误修复之间的大部分差距，占所有修复事件的70%。总之,该系统暴露了DSB修复过程中的通量，解耦感应和修复动力学，并提示高保真修复在限制CRISPR介导的诱变效率方面的重要作用。
