PROTAC molecule-mediated SpCas9 protein degradation for precise genome editing

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

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.

Authors 5

1. Renhong Sun
   Affiliation as printed

   Gluetacs Therapeutics (Shanghai) Co, Ltd, Pudong District, Shanghai, China

   Gluetacs Therapeutics (Shanghai) Co, Ltd

2. Minkang Tan Aachen Department of Cell and Chemical Biology

   Leiden University Medical Center

   Affiliation as printed

   Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands

   Leiden University Medical Center

3. Maarten Kip Aachen Department of Cell and Chemical Biology

   Leiden University Medical Center

   Affiliation as printed

   Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands

   Leiden University Medical Center

4. Xiaobao Yang
   Affiliation as printed

   Gluetacs Therapeutics (Shanghai) Co, Ltd, Pudong District, Shanghai, China

   Gluetacs Therapeutics (Shanghai) Co, Ltd

5. Baoxu Pang corresponding Aachen Department of Cell and Chemical Biology

   Leiden University Medical Center

   Affiliation as printed

   Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands

   Leiden University Medical Center