CRISPR-Cas9 has been developed into a powerful molecular tool for genome engineering, and it has revolutionized the field of biomedical research. Despite the tremendous potential of CRISPR-Cas9 in biomedical research, precise control of CRISPR-Cas9 over the dose and exposure time is important to expand its applications. In this study, we fused Cas9 with a peptide termed small molecule-assisted shut-off (SMASh) consisting of a protease domain and a degron domain derived from hepatitis C virus (HCV). The presence of SMASh allows tight control of the Cas9 stability via a clinically approved HCV protease inhibitor asunaprevir (ASV). We showed that the engineered Cas9 responded to ASV administration and rapidly degraded in a dose- and time-dependent manner. Cas9 degradation was reversible upon ASV removal that restored the gene editing activity. We also showed that limiting the level of Cas9 in cells increased the specificity of gene editing. The SMASh tag therefore provides an effective tool to control Cas9 stability, allowing an improvement in the accuracy, safety, and versatility of the CRISPR-Cas9 system for genome editing and gene regulation studies.

CRISPR-Cas9已发展成为用于基因组工程的强大分子工具，它彻底改变了生物医学研究领域。尽管CRISPR-Cas9在生物医学研究中具有巨大潜力，但精确控制CRISPR-Cas9的剂量和暴露时间对于扩大其应用非常重要。在这项研究中，我们将Cas9与一种称为小分子辅助关闭（SMASh）的肽融合，该肽由蛋白酶结构域和衍生自丙型肝炎病毒（HCV）的degron结构域组成。SMASh的存在允许通过临床批准的HCV蛋白酶抑制剂Asunaprevir（ASV）严格控制Cas9稳定性。我们表明，工程改造的Cas9对ASV给药有反应，并以剂量​​和时间依赖性方式迅速降解。去除ASV后，Cas9降解是可逆的，从而恢复了基因编辑活性。我们还表明，限制细胞中Cas9的水平可提高基因编辑的特异性。因此，SMASh标签提供了一种有效的工具来控制Cas9的稳定性，从而提高了CRISPR-Cas9系统在基因组编辑和基因调控研究中的准确性，安全性和多功能性。