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

  • GENOMICS. TRANSCRIPTOMICS
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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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ACKNOWLEDGMENTS

We would like to thank Dr. Jian Kang, Dr. Deji Luan and Dr. Kang Zhang for the suggestions in performing experiments.

Funding

This work was supported by National Major Project for Production of Transgenic Breeding (no. 2016ZX08007-002) and National Natural Science Foundation of China (no. 31873033).

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Authors and Affiliations

  1. Key Laboratory of Animal Biotechnology, Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China

    P. Deng, X. C. Dong, X. Y. Wang, Y. P. Gao & F. S. Quan

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  2. X. C. Dong
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  4. Y. P. Gao
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Contributions

PD and XCD contributed equally to this work. PD and XCD have made substantial contributions to conception, acquisition of data, drafting the manuscript; XYW have made substantial contributions to analysis and interpretation of data; YPG and FSQ have given final approval of the version to be published. All authors of this article agree to be accountable for all aspects of the work.

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Correspondence to Y. P. Gao or F. S. Quan.

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Deng, P., Dong, X.C., Wang, X.Y. et al. Verification of CRISPR/Cas9 Activity In Vitro via SSA-Based Dual-Luciferase Reporter System. Mol Biol (2024). https://doi.org/10.1134/S0026893324700092

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