PROTAC molecule-mediated SpCas9 protein degradation for precise genome editing

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 resulted in a number of methods to control the function and activity of Cas9 proteins to minimize off-target editing effects. Proteolysis-targeting chimeras (PROTACs) are an emerging technology using the ubiquitin-proteasome system to selectively degrade the target proteins. Here, we developed CASPROTAC 6, a reversible and cell-permeable degrader directly targeting SpCas9 proteins for degradation. The warhead of CASPROTACs was derived by bridging a known SpCas9 binder BRD7087 with an S-substituted Lenalidomide cereblon (CRBN) ligand via the 6-carbon alkyl linker. Our data showed that CASPROTAC 6 is a broad-spectrum degrader of SpCas9 and SpCas9 variants such as dCas9 and Cas9 nickases. The CASPROTAC 6 degraded SpCas9 and the SpCas9-guide-RNA complex via the proteasome system by ∼50%. As a result, the CASPROTAC 6 molecule can reduce the CRISPR/Ca9-mediated genome editing by 30%. The CASPROTAC 6 molecules could be used to regulate the CRISPR/SpCas9-associated nucleases for precise genome editing to reduce off-target effects and strengthen biosafety.