CLEAR Strategy Inhibited HSV Proliferation Using Viral Vectors Delivered CRISPR-Cas9

Herpes simplex virus type 1 (HSV-1) is a leading cause of encephalitis and infectious blindness. The commonly used clinical therapeutic drugs are nucleoside analogues such as acyclovir. However, current drugs for HSV cannot eliminate the latent virus or viral reactivation. Therefore, the development of new treatment strategies against latent HSV has become an urgent need. To comprehensively suppress the proliferation of HSV, we designed the CLEAR strategy (coordinated lifecycle elimination against viral replication). VP16, ICP27, ICP4, and gD-which are crucial genes that perform significant functions in different stages of the HSV infection lifecycle-were selected as targeting sites based on CRISPR-Cas9 editing system. In vitro and in vivo investigations revealed that genome editing by VP16, ICP27, ICP4 or gD single gene targeting could effectively inhibit HSV replication. Moreover, the combined administration method (termed "Cocktail") showed superior effects compared to single gene editing, which resulted in the greatest decrease in viral proliferation. Lentivirus-delivered CRISPR-Cas9/gRNA editing could effectively block HSV replication. The CLEAR strategy may provide new insights into the potential treatment of refractory HSV-1-associated diseases, particularly when conventional approaches have encountered resistance.