A chemical toolbox for labeling and degrading engineered Cas proteins

  • The discovery of clustered regularly interspaced short palindromic repeats and their associated proteins (Cas) has revolutionized the field of genome and epigenome editing. A number of new methods have been developed to precisely control the function and activity of Cas proteins, including fusion proteins and small-molecule modulators. Proteolysis-targeting chimeras (PROTACs) represent a new concept using the ubiquitin-proteasome system to degrade a protein of interest, highlighting the significance of chemically induced protein-E3 ligase interaction in drug discovery. Here, we engineered Cas proteins (Cas9, dCas9, Cas12, and Cas13) by inserting a Phe-Cys-Pro-Phe (FCPF) amino acid sequence (known as the π-clamp system) and demonstrate that the modified CasFCPF proteins can be (1) labeled in live cells by perfluoroaromatics carrying the fluorescein or (2) degraded by a perfluoroaromatics-functionalized PROTAC (PROTAC-FCPF). A proteome-wide analysis of PROTAC-FCPF-mediated Cas9FCPF protein degradation revealed a high target specificity, suggesting a wide range of applications of perfluoroaromatics-induced proximity in the regulation of stability, activity, and functionality of any FCPF-tagging protein.

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Author:Rodrigo Gama BrambilaORCiDGND, Jie Chen, Yasamin DabiriGND, Georg TascherORCiDGND, Václav NěmecORCiD, Christian MünchORCiD, Guangqi SongORCiDGND, Stefan KnappORCiD, Xinlai ChengORCiDGND
Parent Title (English):JACS Au
Publisher:ACS Publications
Place of publication:Washington, DC
Document Type:Article
Date of Publication (online):2021/05/12
Date of first Publication:2021/05/12
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2023/05/04
Tag:CRISPR/Cas9; Cas9 inhibitor; PROTAC; chemical-induced proximity; genome editing; π-clamp
Page Number:9
First Page:777
Last Page:785
We thank DFG for the financial support (CH 1690/2-1 and 2-3). This work was supported in part by the LOEWE Center Frankfurt Cancer Institute funded by the Hessen State Ministry for Higher Education, Research and the Arts [III L 5-519/03/03.001-(0015)].
Institutes:Biochemie, Chemie und Pharmazie / Biochemie und Chemie
Medizin / Medizin
Fachübergreifende Einrichtungen / Buchmann Institut für Molekulare Lebenswissenschaften (BMLS)
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften
5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Licence (German):License LogoCreative Commons - Namensnennung-Nicht kommerziell - Keine Bearbeitung 4.0