Verification of CRISPR/Cas9 Activity In Vitro via SSA-Based Dual-Luciferase Reporter System

IF 1.5 4区生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Molecular Biology Pub Date : 2024-03-17 DOI:10.1134/s0026893324700092

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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.

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

摘要 CRISPR/Cas9 技术已成为精确整合基因组的强大而有前途的工具，可应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导 RNA（sgRNA）。目前有多种方法可用于评估 sgRNA 的裂解活性，其中一种是 T7 内切酶 I（T7EI）检测法，这种方法费时费力，尤其是必须同时处理多个样本时。本研究开发了一种简单快速的方法来检测 sgRNA 的裂解活性。基于单链退火（SSA）修复机制，我们利用萤火虫荧光素酶构建了一个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 sgRNA 的切割效率最高（p < 0.01）。因此，将 T7EI 检测法与本研究建立的方法进行比较，以确定其准确性和灵敏度，两种方法的结果一致，表明 SSA 报告系统与 T7EI 检测法兼容。与需要 PCR 扩增等多个步骤的 T7EI 检测相比，一步转染的 SSA 报告系统可在大约两天内完成大规模 sgRNA 的转染。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 的裂解活性，从而为 CRISPR/Cas9 在基因组编辑中高效选择 sgRNA 提供了一个稳健可靠的过程。

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来源期刊

Molecular Biology 生物-生化与分子生物学

CiteScore

1.30

自引率

8.30%

发文量

审稿时长

3 months

期刊介绍： Molecular Biology is an international peer reviewed journal that covers a wide scope of problems in molecular, cell and computational biology including genomics, proteomics, bioinformatics, molecular virology and immunology, molecular development biology, molecular evolution and related areals. Molecular Biology publishes reviews, experimental and theoretical works. Every year, the journal publishes special issues devoted to most rapidly developing branches of physical-chemical biology and to the most outstanding scientists.