Uncovering the dynamics of precise repair at CRISPR/Cas9-induced double-strand breaks

Daniela Ben-Tov, Fabrizio Mafessoni, Amit Cucuy, Arik Honig, Cathy Melamed-Bessudo () and Avraham A. Levy ()
Additional contact information
Daniela Ben-Tov: Weizmann Institute of Science
Fabrizio Mafessoni: Weizmann Institute of Science
Amit Cucuy: Weizmann Institute of Science
Arik Honig: Weizmann Institute of Science
Cathy Melamed-Bessudo: Weizmann Institute of Science
Avraham A. Levy: Weizmann Institute of Science

Nature Communications, 2024, vol. 15, issue 1, 1-16

Abstract: Abstract CRISPR/Cas9 is widely used for precise mutagenesis through targeted DNA double-strand breaks (DSBs) induction followed by error-prone repair. A better understanding of this process requires measuring the rates of cutting, error-prone, and precise repair, which have remained elusive so far. Here, we present a molecular and computational toolkit for multiplexed quantification of DSB intermediates and repair products by single-molecule sequencing. Using this approach, we characterize the dynamics of DSB induction, processing and repair at endogenous loci along a 72 h time-course in tomato protoplasts. Combining this data with kinetic modeling reveals that indel accumulation is determined by the combined effect of the rates of DSB induction processing of broken ends, and precise versus error repair. In this study, 64–88% of the molecules were cleaved in the three targets analyzed, while indels ranged between 15–41%. Precise repair accounts for most of the gap between cleavage and error repair, representing up to 70% of all repair events. Altogether, this system exposes flux in the DSB repair process, decoupling induction and repair dynamics, and suggesting an essential role of high-fidelity repair in limiting the efficiency of CRISPR-mediated mutagenesis.

Date: 2024
References: View references in EconPapers View complete reference list from CitEc
Citations:

Downloads: (external link)
https://www.nature.com/articles/s41467-024-49410-x Abstract (text/html)

Related works:
This item may be available elsewhere in EconPapers: Search for items with the same title.

Export reference: BibTeX RIS (EndNote, ProCite, RefMan) HTML/Text

Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-49410-x

Ordering information: This journal article can be ordered from
https://www.nature.com/ncomms/

DOI: 10.1038/s41467-024-49410-x

Access Statistics for this article

Nature Communications is currently edited by Nathalie Le Bot, Enda Bergin and Fiona Gillespie

More articles in Nature Communications from Nature
Bibliographic data for series maintained by Sonal Shukla () and Springer Nature Abstracting and Indexing ().