Pamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria, mammalian cells, and mice in a DNA topology-specific manner
Abstract Background Regulation of the target DNA cleavage activity of CRISPR/Cas has naturally evolved in a few bacteria or bacteriophages but is lacking in higher species. Thus, identification of bioactive agents and mechanisms that can suppress the activity of Cas9 is urgently needed to rebalance...
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| Format: | Article |
| Language: | English |
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BMC
2025-03-01
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| Series: | Genome Biology |
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| Online Access: | https://doi.org/10.1186/s13059-025-03521-w |
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| author | Yuxuan Zhang Wentao Zou Yueyang Zhou Jiaqi Chen Youtian Hu Fang Wu |
| author_facet | Yuxuan Zhang Wentao Zou Yueyang Zhou Jiaqi Chen Youtian Hu Fang Wu |
| author_sort | Yuxuan Zhang |
| collection | DOAJ |
| description | Abstract Background Regulation of the target DNA cleavage activity of CRISPR/Cas has naturally evolved in a few bacteria or bacteriophages but is lacking in higher species. Thus, identification of bioactive agents and mechanisms that can suppress the activity of Cas9 is urgently needed to rebalance this new genetic pressure. Results Here, we identify four specific inhibitors of Cas9 by screening 4607 compounds that could inhibit the endonuclease activity of Cas9 via three distinct mechanisms: substrate-competitive and protospacer adjacent motif (PAM)-binding site-occupation; substrate-targeting; and sgRNA-targeting mechanisms. These inhibitors inhibit, in a dose-dependent manner, the activity of Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9), and SpyCas9 nickase-based BE4 base editors in in vitro purified enzyme assays, bacteria, mammalian cells, and mice. Importantly, pamoic acid and carbenoxolone show DNA-topology selectivity and preferentially inhibit the cleavage of linear DNA compared with a supercoiled plasmid. Conclusions These pharmacologically selective inhibitors and new mechanisms offer new tools for controlling the DNA-topology selective activity of Cas9. |
| format | Article |
| id | doaj-art-daf715ab31974ba4bd2df6ac2a0efbf8 |
| institution | Kabale University |
| issn | 1474-760X |
| language | English |
| publishDate | 2025-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Genome Biology |
| spelling | doaj-art-daf715ab31974ba4bd2df6ac2a0efbf82025-08-20T03:40:44ZengBMCGenome Biology1474-760X2025-03-0126113810.1186/s13059-025-03521-wPamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria, mammalian cells, and mice in a DNA topology-specific mannerYuxuan Zhang0Wentao Zou1Yueyang Zhou2Jiaqi Chen3Youtian Hu4Fang Wu5Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong UniversityKey Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong UniversityKey Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong UniversityKey Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong UniversityKey Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong UniversityKey Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong UniversityAbstract Background Regulation of the target DNA cleavage activity of CRISPR/Cas has naturally evolved in a few bacteria or bacteriophages but is lacking in higher species. Thus, identification of bioactive agents and mechanisms that can suppress the activity of Cas9 is urgently needed to rebalance this new genetic pressure. Results Here, we identify four specific inhibitors of Cas9 by screening 4607 compounds that could inhibit the endonuclease activity of Cas9 via three distinct mechanisms: substrate-competitive and protospacer adjacent motif (PAM)-binding site-occupation; substrate-targeting; and sgRNA-targeting mechanisms. These inhibitors inhibit, in a dose-dependent manner, the activity of Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9), and SpyCas9 nickase-based BE4 base editors in in vitro purified enzyme assays, bacteria, mammalian cells, and mice. Importantly, pamoic acid and carbenoxolone show DNA-topology selectivity and preferentially inhibit the cleavage of linear DNA compared with a supercoiled plasmid. Conclusions These pharmacologically selective inhibitors and new mechanisms offer new tools for controlling the DNA-topology selective activity of Cas9.https://doi.org/10.1186/s13059-025-03521-wAnti-CRISPRSelective small-molecule inhibitorsMode of actionDNA topologyMice model of hydrodynamic injection |
| spellingShingle | Yuxuan Zhang Wentao Zou Yueyang Zhou Jiaqi Chen Youtian Hu Fang Wu Pamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria, mammalian cells, and mice in a DNA topology-specific manner Genome Biology Anti-CRISPR Selective small-molecule inhibitors Mode of action DNA topology Mice model of hydrodynamic injection |
| title | Pamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria, mammalian cells, and mice in a DNA topology-specific manner |
| title_full | Pamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria, mammalian cells, and mice in a DNA topology-specific manner |
| title_fullStr | Pamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria, mammalian cells, and mice in a DNA topology-specific manner |
| title_full_unstemmed | Pamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria, mammalian cells, and mice in a DNA topology-specific manner |
| title_short | Pamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria, mammalian cells, and mice in a DNA topology-specific manner |
| title_sort | pamoic acid and carbenoxolone specifically inhibit crispr cas9 in bacteria mammalian cells and mice in a dna topology specific manner |
| topic | Anti-CRISPR Selective small-molecule inhibitors Mode of action DNA topology Mice model of hydrodynamic injection |
| url | https://doi.org/10.1186/s13059-025-03521-w |
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