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

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 sgRN...

Full description

Saved in:
Bibliographic Details
Published in:Molecular biology (New York) 2024, Vol.58 (3), p.461-470
Main Authors: Deng, P., Dong, X. C., Wang, X. Y., Gao, Y. P., Quan, F. S.
Format: Article
Language:English
Subjects:
Biochemistry
Biomedical and Life Sciences
CRISPR
Efficiency
Endonuclease
Genome editing
Genomics. Transcriptomics
Human Genetics
Life Sciences
Transfection
Citations: Items that this one cites
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0026-8933
1608-3245
DOI:10.1134/S0026893324700092