Abstract

The CRISPR/Cas9 technique has emerged as a powerful and promising tool for precise genomic integration, which applied to various cell types and organisms, but its efficiency largely depends on single-guide RNA (sgRNA). There are multiple strategies available to evaluate the cleavage activity of sgRNAs, and one such approach is T7 endonuclease I (T7EI) assay, which is laborious and time consuming, especially when one must address multiple samples in parallel. In this study, a simple and rapid method to detect the cleavage activity of sgRNA was developed. Based on the single-strand annealing (SSA) repair mechanism, a surrogate reporter system using firefly luciferase was constructed to evaluate the targeting efficiency of sgRNAs. Using this system, the luciferase activities of eight sgRNAs were observed, and one of them had highest cutting efficiency (p < 0.01). Thereby, T7EI assay was compared with the method established in this study to determine the accuracy and sensitivity, and the results of these two methods were consistent suggesting that the SSA reporter system was compatible with T7EI assay. Compared with T7EI assay requiring multiple steps, such as PCR amplification, the SSA reporter system with one-step transfection can be completed on a large scale of sgRNAs within approximate two days. These findings suggested that SSA-based reporter system can accurately and rapidly evaluate the cleavage activities of multiple sgRNAs, thereby providing a robust and reliable process for CRISPR/Cas9 to select sgRNAs efficiently in genome editing.

中文翻译：

---

通过基于 SSA 的双荧光素酶报告系统验证 CRISPR/Cas9 体外活性

摘要

CRISPR/Cas9技术已成为一种强大且有前途的基因组精确整合工具，适用于各种细胞类型和生物体，但其效率在很大程度上取决于单向导RNA（sgRNA）。有多种策略可用于评估 sgRNA 的切割活性，其中一种方法是 T7 核酸内切酶 I (T7EI) 测定，该方法费力且耗时，尤其是当必须并行处理多个样品时。本研究开发了一种简单快速的方法来检测 sgRNA 的切割活性。基于单链退火（SSA）修复机制，构建了使用萤火虫荧光素酶的替代报告系统来评估sgRNA的靶向效率。利用该系统，观察了8种sgRNA的荧光素酶活性，其中一种具有最高的切割效率（p <0.01）。因此，将T7EI测定法与本研究建立的方法进行比较以确定准确性和灵敏度，两种方法的结果一致，表明SSA报告系统与T7EI测定法兼容。与需要PCR扩增等多个步骤的T7EI检测相比，一步转染的SSA报告系统可以在大约两天内完成大规模sgRNA的检测。这些发现表明，基于SSA的报告系统可以准确、快速地评估多个sgRNA的切割活性，从而为CRISPR/Cas9在基因组编辑中有效选择sgRNA提供稳健可靠的过程。