| Dublin Core |
PKP Metadata Items |
Metadata for this Document |
| 1. |
Title |
Title of document |
Novel CRISPR/Cas9-Based Approaches for Quantitative Study of DSB Repair Mechanics |
| 2. |
Creator |
Author's name, affiliation, country |
A. V. Smirnov; Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences; Russian Federation |
| 2. |
Creator |
Author's name, affiliation, country |
A. M. Yunusova; Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences; Russian Federation |
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Subject |
Discipline(s) |
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| 3. |
Subject |
Keyword(s) |
CRISPR/Cas9; double-strand break (DSB) repair; genetic reporter |
| 4. |
Description |
Abstract |
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. 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. |
| 5. |
Publisher |
Organizing agency, location |
The Russian Academy of Sciences |
| 6. |
Contributor |
Sponsor(s) |
Russian Science Foundation (22-74-00084)
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| 7. |
Date |
(DD-MM-YYYY) |
04.09.2025
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| 8. |
Type |
Status & genre |
Peer-reviewed Article |
| 8. |
Type |
Type |
Review Article |
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Format |
File format |
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| 10. |
Identifier |
Uniform Resource Identifier |
https://kazanmedjournal.ru/0320-9725/article/view/685801 |
| 10. |
Identifier |
Digital Object Identifier (DOI) |
10.31857/S0320972525040017 |
| 10. |
Identifier |
eLIBRARY Document Number (EDN) |
IHSTQC |
| 11. |
Source |
Title; vol., no. (year) |
Biohimiâ; Vol 90, No 4 (2025) |
| 12. |
Language |
English=en |
ru |
| 13. |
Relation |
Supp. Files |
Fig. 1. Fluorescent genetic reporters of DSBs. a - Traffic light reporter (Traffic light reporter). Challenged by Cas9, DSB is repaired either through HDR using a GFP donor sequence or through NHEJ/MMEJ, which lead to reading frame shift and mCherry activation [27]. b - Repair-seq applies high-throughput gene repair gene knockdown using dCas9-KRAB and the CRISPRi gRNA library. After integration of viruses into the genome and knockdown of target genes, the transduced cell population is electroporated with Cas9 ribonucleoprotein complex (RNP) and gRNA against a target site adjacent to the CRISPRi gRNA. This allows us to assess the contribution of the silenced gene to DSB repair by analysing Cas9 indels [25]. Colours indicate unique gRNAs and cell clones expressing them (not related to the fluorescence signal) (381KB)
Fig. 2. New CRISPR/Cas9-based quantitative DSB reporters. a - DSB-TRIP uses short transposon sequences embedded at random locations in the genome. The transposons contain a 16-nucleotide barcode paired with a target site for Cas9, allowing the barcode and indels to be sequenced together (1) and associated with different chromatin contexts after transposon localisation (2). The method allows us to distinguish between repair pathways such as NHEJ, MMEJ and single stranded template based repair (SSTR), which has similar initial steps to homologous recombination (HR) [19]. b - The ddXR method combines the induction of deletions with Cas9 and their detection with ddPCR. The ddXR allows the determination of deletion and inversion frequencies using the same probe [20]. c - Concatemer sequencing is based on microinjection of linearised DNA into the pronucleus, where the DNA remains bound to Cas9 after cutting (reaction with thermally inactivated protein is used as a control). Barcodes allow identification of single copies and investigation of DSB repair pathways by sequencing of fusion sites between copies (407KB)
Fig. 3. Features of the CRISPR/Cas9 system that may affect the outcome of DSB repair. The binding time of Cas9 and DNA is determined by unknown cellular factors. Cas9 indel signatures depend on the nature of the DNA ends after cutting [66]. Cas9 protein domains affect DSB repair locally or allow switching of Cas9 activity during the cell cycle. Inducible Cas9 variants control the timing of DSB emergence by photoactivation (very fast CRISPR) or chemical stabilisation (DD-Cas9); DD, destabilising domain; PAM, motif near the protospacer (317KB)
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Coverage |
Geo-spatial location, chronological period, research sample (gender, age, etc.) |
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| 15. |
Rights |
Copyright and permissions |
Copyright (c) 2025 Russian Academy of Sciences
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