建立基于裂解的单质粒双荧光素酶替代报告物,用于评估 CRISPR-Cas12a 系统的裂解效率及其主要应用。

IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY CRISPR Journal Pub Date : 2024-06-01 DOI:10.1089/crispr.2024.0038
Yaoqiang Shi, Qi Tan, Chunhui Yang, Shilin Li, Yujia Li, Baoren He, He Xie, Xiaoqiong Duan, Limin Chen
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引用次数: 0

摘要

CRISPR-Cas 技术是一种广泛使用的基因编辑工具,包括 gRNA 引导的序列识别和 Cas 核酸酶介导的裂解。设计和评估 gRNA 对提高 CRISPR/Cas 编辑效率至关重要。单链退火、体外裂解和 T7 内切酶 I(T7EI)等各种检测方法通常用于评估 gRNA 介导的 Cas 蛋白裂解活性。本研究构建了萤火虫荧光素酶和雷尼拉荧光素酶共表达和基于裂解的单质粒双荧光素酶替代报告物,以评估 gRNA 介导的 Cas12a 裂解效率。CRISPR-Cas12a的裂解活性可以通过萤火虫荧光素酶活性的恢复程度来定量测定。CRISPR-Cas12a的裂解效率可以通过萤火虫荧光素酶活性的恢复程度来定量测定。利用该系统评估了 CRISPR-Cas12a 对乙型肝炎病毒(HBV)/D 表达质粒的裂解效率,结果显示 gRNA 的裂解效率与使用酶联免疫吸附测定法测量的 HBV 基因表达之间存在负相关。这种简单、高效、可量化的系统只需要双荧光素酶载体和CRISPR-Cas12a载体,因此是选择有效基因编辑gRNA的重要工具。
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Establishment of a Cleavage-Based Single-Plasmid Dual-Luciferase Surrogate Reporter for the Cleavage Efficiency Evaluation of CRISPR-Cas12a Systems and Its Primary Application.

CRISPR-Cas technology is a widely utilized gene-editing tool that involves gRNA-guided sequence recognition and Cas nuclease-mediated cleavage. The design and evaluation of gRNA are essential for enhancing CRISPR/Cas editing efficiency. Various assays such as single-strand annealing, in vitro cleavage, and T7 endonuclease I (T7EI) are commonly used to assess gRNA-mediated Cas protein cleavage activity. In this study, a firefly luciferase and Renilla luciferase co-expressed and a cleavage-based single-plasmid dual-luciferase surrogate reporter was built to evaluate the gRNA-mediated Cas12a cleavage efficiency. The cleavage activities of CRISPR-Cas12a can be quantitatively determined by the recovery degree of firefly luciferase activity. The cleavage efficiency of CRISPR-Cas12a can be quantitatively measured by the recovery of firefly luciferase activity. By using this system, the cleavage efficiency of CRISPR-Cas12a on hepatitis B virus (HBV)/D expression plasmid was evaluated, revealing a negative correlation between gRNA cleavage efficiency and HBV gene expression measured using an enzyme-linked immunosorbent assay. This simple, efficient, and quantifiable system only requires the dual-luciferase vector and CRISPR-Cas12a vector, making it a valuable tool for selecting effective gRNAs for gene editing.

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来源期刊
CRISPR Journal
CRISPR Journal Biochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍: In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR. Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.
期刊最新文献
Early Detection of Wildlife Disease Pathogens Using CRISPR-Cas System Methods. CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans. Genome Editing in Apicomplexan Parasites: Current Status, Challenges, and Future Possibilities. Mutation-Specific CRISPR Targeting with SaCas9 and AsCas12a Restores Therapeutic Sensitivity in Treatment-Resistant Melanoma. Characterization of Research Support of Genome Editing Technologies and Transition to Clinical Trials.
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