Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9
Yili Feng1,2,3, Sicheng Liu4,5, Ruodan Chen4,6,5
1Innovation Center for Minimally Invasive Technique and Device, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310019, China. eric_feng@zju.edu.cn.
Abstract:
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.
Insights
CRISPR/Cas9 genome editing efficiency depends on Cas9-sgRNA binding. Variations in binding affinity and residence time influence DNA double-strand break repair, leading to diverse mutations and offering opportunities for optimizing CRISPR technology.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a powerful tool for genome editing.
- CRISPR/Cas9 efficiency relies on Cas9-single-guide RNA (sgRNA) binding affinity and residence time.
- The impact of binding dynamics on DNA double-strand break (DSB) repair outcomes is underexplored.
Purpose of the Study:
- To investigate how Cas9-sgRNA target binding affinity and residence duration affect DSB repair pathway choice in CRISPR/Cas9 genome editing.
- To understand the mechanisms leading to heterogeneous mutations after CRISPR/Cas9-induced DSBs.
- To identify strategies for optimizing CRISPR/Cas9 technology based on binding dynamics.
Main Methods:
- The study proposes a theoretical framework to analyze the relationship between Cas9-sgRNA binding kinetics and DSB repair.
- It discusses the potential influence of varying binding affinities and residence times on non-homologous end joining (NHEJ) versus homology-directed repair (HDR).
- The authors review existing literature and propose experimental approaches to validate their hypothesis.
Main Results:
- Cas9-sgRNA binding affinity and residence time at the target site can influence the selection of DSB repair pathways.
- Variations in binding dynamics contribute to the heterogeneity of mutations observed in CRISPR/Cas9 genome editing outcomes.
- Understanding these dynamics provides insights into controlling editing precision and outcomes.
Conclusions:
- The binding characteristics of Cas9-sgRNA complexes play a critical role in determining the fidelity and mutation profile of CRISPR/Cas9 editing.
- Modulating Cas9-sgRNA binding affinity and residence time presents a novel strategy for enhancing the precision and predictability of genome editing.
- This research opens avenues for optimizing CRISPR/Cas9 applications in research and clinical settings.
Related Concept Videos
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01:28CRISPR/Cas9 Genome Editing
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01:59CRISPR
02:04Fixing Double-strand Breaks
02:04Fixing Double-strand Breaks