PROTAC molecule-mediated SpCas9 protein degradation for precise genome editing

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Abstract CRISPR (clustered regularly interspaced short palindromic repeats) technology has revolutionized both fundamental research and genomic medicine. The need for precise control of SpCas9-based CRISPR genome editing has led to the development of various strategies to regulate Cas9 activity, aiming to minimize off-target effects. Proteolysis-targeting chimeras (PROTACs) use the ubiquitin-proteasome system to selectively degrade target proteins. Here, we developed CASPROTAC 6, a reversible and cell-permeable PROTAC degrader that directly targets SpCas9 proteins for degradation. The CASPROTAC 6 molecule was derived by linking a known SpCas9 binder BRD7087 with an S-substituted Lenalidomide cereblon (CRBN) ligand via a six-carbon alkyl linker. Our data show that CASPROTAC 6 is a broad-spectrum degrader of SpCas9 and variants such as dCas9 and Cas9 nickases. CASPROTAC 6 degrades SpCas9 and the SpCas9-guide RNA complex via the proteasome system by approximately 50%. As a result, CASPROTAC 6 effectively enhanced the precision of CRISPR/Cas9-mediated genome editing by increasing target specificity. CASPROTAC 6 may serve as modulators of CRISPR/SpCas9-associated nucleases, enabling precise genome editing while reducing off-target effects and enhancing biosafety.

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