CRISPR/Cas9 is a type of endonuclease that relies on sgRNA and can recognize target DNA sequences complementary to sgRNA and cut or edit them. It was first used in in vitro DNA editing experiments in 2012 [1] and was awarded the Nobel Prize in Chemistry in 2020. CRISPR/Cas9 genome editing technology has been approved by the US FDA in 2023 for the treatment of sickle cell disease and thalassemia（ https://clinicaltrials.gov/ ）. At present, it is used to treat viral keratitis, tumors HIV、 More than ten diseases, including drug-resistant bacterial infections, are also undergoing clinical trials. However, CRISPR/Cas9 has been found to have off target effects during gene editing in research and clinical practice, and the redundant activity of Cas9 can also cause safety issues such as genotoxicity [2-3]. Therefore, the development of inhibitors or drug leads that can control redundant CRISPR/Cas9 genome editing activity and reduce its off target effects is an urgent need in current genomic pharmacology research.
Recently, a team led by Wu Fang from Shanghai Jiao Tong University published an online article titled "Pamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria" in the journal Genome Biology, The original research paper on mammalian cells and mice in a DNA topology specific manner discovered the first selective targeted inhibitor that can inhibit Cas9 activity both in vitro and in vivo, providing a new tool and drug lead for the research and application of genome editing technology.
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This study established a large-scale inhibitor screening model based on the in vitro endonuclease activity of Cas9. Four Cas9 selective inhibitors were screened and discovered from 4607 FDA approved drugs or natural products. It can selectively inhibit the activity of Type II Cas9 without inhibiting the activity of Type V CRISPR/Cas12. Through biochemical and other methods, research has revealed that four inhibitors can inhibit Cas9 activity through three molecular mechanisms: competitive occupation of PAM sites, targeting of sgRNA, and targeting of substrate DNA. Among them, PAM site inhibitors pamoic acid and carbenoxolone can selectively inhibit the cleavage of SpyCas9 on DNA substrates with different topological structures. They have a better inhibitory effect on SpyCas9's cleavage of linear DNA substrates, but a poorer inhibitory effect on circular substrates. The difference in IC50 values between the two can be as high as 10-20 times, indicating that Cas9 may have different affinities or molecular mechanisms for recognizing DNA substrates with different topological structures.
In order to further investigate the inhibitory effect of inhibitors on Cas9 genome editing activity and their regulation of off target effects, the author team has established multiple methods and platforms for studying CRISPR/Cas9 editing activity. These methods and platforms can investigate the inhibitory effect of inhibitors on Cas9 gene editing activity in prokaryotic bacteria and eukaryotic cells under two mechanisms: non homologous terminal junction repair (NHEJ) and homologous mediated repair (HDR). In addition, the author team also took the lead in establishing an animal model for studying the regulation of Cas9 genome editing activity by inhibitors in vivo. Research has found that pamoic acid, carbenoxolone, and dalbavancin (targeting sgRNA mechanism) can dose dependently inhibit the activity of gene editors such as SpyCas9, SauCas9, and BE4 in bacteria and cells, and significantly reduce their off target effects. Meanwhile, pamoic acid and carbenoxolone can significantly inhibit the editing of PCSK9 gene loci in liver tissue cells by SpyCas9 and the resulting loss of large gene fragments in mice, maintaining genomic stability in vivo. Pamoic acid and carbenoxolone are the first in vivo inhibitors of Cas9 with pharmacological activity and good in vivo safety. This result can provide a new research tool for the study and application of Cas9.
In summary, this study identified four Cas9 selective inhibitors and revealed three different inhibition mechanisms: competitive occupation of PAM sites, targeting of sgRNA, and targeting of substrate DNA. These inhibitors not only regulate the genome editing activity of Cas9 in pure enzymes, bacteria, cells, and mice in vitro, but also provide a new perspective for elucidating the substrate topology selectivity mechanism of Cas9. They can also provide small molecule inhibitor tools for regulating Cas9's in vitro and in vivo off target effects and genotoxicity. At present, relevant inhibitors and their applications have also applied for Chinese invention patents.
Zhang Yuxuan, a doctoral student at the Institute of Systems Biomedical Research, Shanghai Jiao Tong University, is the first author of the paper, and Researcher Wu Fang is the corresponding author of the paper. Master's and doctoral students Zou Wentao, Chen Jiaqi, Zhou Yueyang, and Hu Youtian also made important contributions to this research.
Original link:
https://genomebiology.biomedcentral.com/articles/10.1186/s13059-025-03521-w
Reference:
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Jinek M, et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–21.
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Frati G, et al., Safety and efficacy studies of CRISPR-Cas9 treatment of sickle cell disease highlights disease-specific responses. Molecular Therapy. 2024;32:4337-4352.
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Nahmad, AD. et al. Frequent aneuploidy in primary human T cells after CRISPR–Cas9 cleavage. Nat. Biotechnol. 2022;40:1807–1813.