Journal of Zhejiang University. Science. BJan 15, 2021

How DNA binding time affects repair pathway choice during CRISPR/Cas9 genome editing

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jan 13, 2026

Abstract

The binding affinity and residence duration of Cas9-sgRNA at target sites may significantly influence DNA repair outcomes.

Variation in target binding affinity and residence time could affect the choice of DNA repair pathway after Cas9-induced double-strand breaks.
Different repair pathways may lead to heterogeneous mutations in genome editing.
Optimizing binding affinity and residence duration may enhance the precision of CRISPR/Cas9 technology.

Simplified

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is widely used for targeted genomic and epigenomic modifications and imaging in cells and organisms, and holds tremendous promise in clinical applications. The efficiency and accuracy of the technology are partly determined by the target binding affinity and residence time of Cas9-single-guide RNA (sgRNA) at a given site. However, little attention has been paid to the effect of target binding affinity and residence duration on the repair of Cas9-induced DNA double-strand breaks (DSBs). We propose that the choice of DSB repair pathway may be altered by variation in the binding affinity and residence duration of Cas9-sgRNA at the cleaved target, contributing to significantly heterogeneous mutations in CRISPR/Cas9 genome editing. Here, we discuss the effect of Cas9-sgRNA target binding and residence on the choice of DSB repair pathway in CRISPR/Cas9 genome editing, and the opportunity this presents to optimize Cas9-based technology.

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How DNA binding time affects repair pathway choice during CRISPR/Cas9 genome editing

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