REVIEW: Novel CRISPR/Cas9-Based Approaches for Quantitative Study of DSB Repair Mechanics

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Alexander V. Smirnov^1,a*, Anastasia M. Yunusova¹

¹Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia

^* To whom correspondence should be addressed.

Received: April 20, 2024; Revised: March 7, 2025; Accepted: April 2, 2025

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This review examines modern approaches to studying double-strand break (DSB) DNA repair in mammalian cells, employing the CRISPR/Cas9 system. Due to its flexibility and efficacy, the Cas9 nuclease is used in numerous genetic reporters. We discuss various fluorescence-based genetic reporters used to monitor the repair process. In addition, among the innovative Cas9-based methods, special attention is given to the techniques that examine both single and multiple DSBs, including approaches such as DSB-TRIP and ddXR. These methods open new possibilities for investigating structural rearrangements or analyzing random genomic sites. Additionally, the review considers how DSBs induced by Cas9 differ from those made by other nucleases and how these peculiarities could impact DNA repair mechanisms. Understanding these differences is crucial for planning experiments aimed at studying DSB repair.

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KEY WORDS: CRISPR/Cas9, double-strand break (DSB) repair, genetic reporter

DOI: 10.1134/S0006297924601813

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