Disruption of Human Papillomavirus 16 E6/E7 Genes Using All-in-One Adenovirus Vectors Expressing Eight Double-Nicking Guide RNAs.

Human papillomavirus (HPV) is a prime target for genome-editing therapy as its E6 and E7 oncogenes are crucial for cancer development and maintenance. A key challenge in CRISPR/Cas9 therapy is the off-target effects. This study utilized a double-nicking technique to introduce DNA breaks in the E6 and E7 regions of HPV16. From 146 gRNA candidates, 16 double-nicking pairs were selected. Multiple combinations of double-nicking (DN)-gRNA pairs were delivered to HPV16-positive cells via lentiviruses, followed by Cas9 nickase (Cas9n) expression. Combinations of 3-4 DN-gRNA pairs effectively killed HPV16-positive cells while sparing HPV-negative cells. Off-target effects were reduced by nearly three orders of magnitude. An "all-in-one" adenovirus (AdV) system expressing four gRNA pairs and Cas9n showed promise in inhibiting tumor growth in HPV16-positive cancer models, demonstrating its potential as a safe and effective treatment for HPV-induced tumors.