新型DNA双链断裂测序方法及其在基因编辑工具酶评估中的应用

DNA double-stranded breaks (DSBs) greatly threaten genome stability and can lead to cell death or tumorigenesis. However, genome editing tool enzymes generate gene deletions, insertions, and replacement with the purpose of gene therapy by inducing DSBs at targeted sites in the genome. Mapping DSB sites is the key for investigation into the mechanism how DNA damage contributes to tumors. Moreover, the efficiency and specificity to generate DSBs also help evaluate various genome editing tool enzymes. Several DSB mapping methods have been developed in recent years to solve the “key step” between DNA damage and tumorigenesis. However, these assays were not suitable for studying complex process of DNA damage and comparing editing efficiency of different engineered enzymes with regards to the absence of accurate quantification. Here we propose to develop a new quantitative DSB-mapping method to determinate different causes of DNA damages in the same cell populations by introducing “molecular barcode” into current assays. We will apply our new assay to evaluating given genome editing tool enzymes by identifying “off-target” sites and estimating the cutting efficiency at “on-target” sites in the same cell populations. In this regard, we will provide a rapid and low-cost solution for pre-application assessment of genome editing tool enzymes.

DNA双链断裂严重威胁遗传信息传递的连续性，可造成细胞死亡或引发癌症。但基因编辑工具酶正是通过在基因组上产生DNA双链断裂的方式来引发基因的缺失、插入和替换等以实现基因治疗的目的。鉴定DNA双链断裂的发生位点是研究DNA损伤与癌症的发病机理之间联系的关键一环，而DNA双链断裂的产生效率及专一性也能被应用于评估基因编辑工具酶。最近几年有多种鉴定DNA双链断裂位点的高通量测序方法问世，但是这些方法都不能定量分析，因而不能用来研究一些较为复杂的DNA损伤过程及比较不同工具酶基因编辑的效率。在本项目中，我们将通过给每个DNA片段添加“分子条码”的方法，在已有的方法的基础上发展出一种新的DNA双链断裂鉴定方法以正确区分复杂DNA损伤过程中引发DNA断裂的各种原因。我们将用新方法鉴定基因编辑工具酶脱靶位点并评估这些酶在靶位点的切割效率，从而为基因编辑工具酶的应用前评估中提供一种快速而低成本的解决方法。

基因编辑工具发挥作用主要是在目标位点上先产生DNA双链断裂，然后进行修复，从而达到目的编辑的效果。真核细胞内的DNA修复可以产生多种产物及副产物，对这些产物的分析可以促进对基因编辑的深入研究，从而寻找更为安全的基因编辑方案。因此，开发一种可以全面检测基因编辑产物的方法不仅可以用于优化基因编辑，还可以促进DNA修复领域的研究。这里，借助于分子条码技术，项目组开发了一个可以全面检测基因编辑产物的方法──PEM-seq。运用这个方法，项目组评估了CRISPR-Cas9及其各种高保真和宽域编辑变体的编辑效果及安全性。并且，项目组还开发了一个全新的CRISPR-Cas9优化体，可以极大地降低基因编辑过程中的染色体易位。该变体具有可见的非常巨大的应用前景。