Identification and Analysis of Small Molecule Inhibitors of CRISPR-Cas9 in Human Cells
Yue Yang,
Donghua Li,
Fen Wan,
Bohong Chen,
Guanglan Wu,
Feng Li,
Yanliang Ren,
Puping Liang,
Jian Wan,
Zhou Songyang
Affiliations
Yue Yang
MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
Donghua Li
MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
Fen Wan
International Cooperation Base of Pesticide and Green Synthesis (Hubei), Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, Department of Chemistry, Central China Normal University, Wuhan 430079, China
Bohong Chen
MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
Guanglan Wu
MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
Feng Li
MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
Yanliang Ren
International Cooperation Base of Pesticide and Green Synthesis (Hubei), Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, Department of Chemistry, Central China Normal University, Wuhan 430079, China
Puping Liang
MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
Jian Wan
International Cooperation Base of Pesticide and Green Synthesis (Hubei), Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, Department of Chemistry, Central China Normal University, Wuhan 430079, China
Zhou Songyang
MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
Genome editing tools based on CRISPR–Cas systems can repair genetic mutations in situ; however, off-target effects and DNA damage lesions that result from genome editing remain major roadblocks to its full clinical implementation. Protein and chemical inhibitors of CRISPR–Cas systems may reduce off-target effects and DNA damage. Here we describe the identification of several lead chemical inhibitors that could specifically inhibit the activity of Streptococcus pyogenes Cas9 (SpCas9). In addition, we obtained derivatives of lead inhibitors that could penetrate the cell membrane and inhibit SpCas9 in cellulo. Two of these compounds, SP2 and SP24, were able to improve the specificity of SpCas9 in cellulo at low-micromolar concentration. Furthermore, microscale thermophoresis (MST) assays showed that SP24 might inhibit SpCas9 activity by interacting with both the SpCas9 protein and the SpCas9–gRNA ribonucleoprotein complex. Taken together, SP24 is a novel chemical inhibitor of SpCas9 which has the potential to enhance therapies that utilize SpCas9.
