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.
摘要
CRISPR/Cas9技术广泛应用于靶向基因编辑、表观遗传学修饰和细胞成像等多个领域,临床应用潜能巨大。然而,Cas9-sgRNA复合物靶点结合强度与滞留时间长短是否会对Cas9诱导的DNA双链断裂(DSB)修复产生影响并不清楚,这个问题也常被忽视。我们先前的研究发现,CRISPR/Cas9技术的有效性和精准性部分取决于Cas9-sgRNA在靶点的结合以及滞留,其靶点结合亲和力和滞留时间会随着靶点不同而不同,从而影响DSB修复途径的选择,这也是CRISPR/Cas9基因编辑异质性产生的一个重要原因。在本文中,我们将讨论CRISPR/Cas9基因编辑中Cas9-sgRNA的靶点结合与滞留如何影响细胞内DSB修复途径的选择,在此基础上提出优化CRISPR/Cas9技术的可能方式。
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08 April 2021
The typesetting format of the online version of the first issue (2021 22(01)) of Journal of Zhejiang University-SCIENCE B is different from that of the printed version (but all the text, figure and table contents in the article are correct). This is due to the new typesetting company adopted this year.
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Acknowledgments
The research is supported by the National Natural Science Foundation of China (Nos. 31671385 and 31870806), the Zhejiang Provincial Natural Science Foundation of China (Nos. LY18C050001 and LQ20C050004), and the Fundamental Research Funds for the Central Universities in China (No. 2019QNA7031). We thank members of the XIE’s lab for their contributions and helpful discussions. We sincerely apologize to authors who are not quoted in references due to space limitations.
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Anyong XIE, Yili FENG, and Sicheng LIU wrote the manuscript. Yili FENG drew the figures. Anyong XIE, Yili FENG, Sicheng LIU, and Ruodan CHEN edited the manuscript. All authors have read and approved the final manuscript.
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Yili FENG, Sicheng LIU, Ruodan CHEN, and Anyong XIE declare that they have no conflicts of interest.
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Feng, Y., Liu, S., Chen, R. et al. Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing. J. Zhejiang Univ. Sci. B 22, 73–86 (2021). https://doi.org/10.1631/jzus.B2000282
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DOI: https://doi.org/10.1631/jzus.B2000282
Key words
- CRISPR/Cas9 genome editing
- Double-strand break (DSB) repair pathway choice
- Target binding affinity
- Target residence