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Hitchhiking of Cas9 with nucleus-localized proteins impairs its controllability and leads to efficient genome editing of NLS-free Cas9.
- Source :
-
Molecular therapy : the journal of the American Society of Gene Therapy [Mol Ther] 2024 Apr 03; Vol. 32 (4), pp. 920-934. Date of Electronic Publication: 2024 Feb 09. - Publication Year :
- 2024
-
Abstract
- CRISPR-Cas9 is the most commonly used genome-editing tool in eukaryotic cells. To modulate Cas9 entry into the nucleus to enable control of genome editing, we constructed a light-controlled CRISPR-Cas9 system to control exposure of the Cas9 protein nuclear localization signal (NLS). Although blue-light irradiation was found to effectively control the entry of Cas9 protein into the nucleus with confocal microscopy observation, effective gene editing occurred in controls with next-generation sequencing analysis. To further clarify this phenomenon, a CRISPR-Cas9 editing system without the NLS and a CRISPR-Cas9 editing system containing a nuclear export signal were also constructed. Interestingly, both Cas9 proteins could achieve effective editing of target sites with significantly reduced off-target effects. Thus, we speculated that other factors might mediate Cas9 entry into the nucleus. However, NLS-free Cas9 was found to produce effective target gene editing even following inhibition of cell mitosis to prevent nuclear import caused by nuclear membrane disassembly. Furthermore, multiple nucleus-localized proteins were found to interact with Cas9, which could mediate the "hitchhiking" of NLS-free Cas9 into the nucleus. These findings will inform future attempts to construct controllable gene-editing systems and provide new insights into the evolution of the nucleus and compatible protein functions.<br />Competing Interests: Declaration of interests W.Z. and H.S. are employees of Guangdong Pharmaceutical University. C.G., Y.J., Haozheng Wang, Z.L., Hui Wang, X.L., M.L., and Y.W. are graduate students of W.Z.<br /> (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)
Details
- Language :
- English
- ISSN :
- 1525-0024
- Volume :
- 32
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- Molecular therapy : the journal of the American Society of Gene Therapy
- Publication Type :
- Academic Journal
- Accession number :
- 38341611
- Full Text :
- https://doi.org/10.1016/j.ymthe.2024.02.008