Precision engineering of human cytomegalovirus without BAC constraints: a Sendai virus-delivered CRISPR/Cas9 approach.

Human cytomegalovirus (HCMV) genetic manipulation traditionally relies on bacterial artificial chromosome (BAC) recombineering, necessitated by its large ~236 kb genome. This approach is limited by the scarcity of HCMV strains engineered into BACs and often requires the deletion of 'non-essential' genes to accommodate the BAC cassette. We developed a novel approach using temperature-sensitive Sendai virus (SeV) vectors to deliver CRISPR/Cas9 for targeted HCMV genome editing without these constraints. This system achieves high editing efficiency (80-90%) in fibroblasts, epithelial cells and endothelial cells without BAC intermediates. As proof of principle, we targeted the HCMV (TB40/E strain) pentamer complex (PC) genes UL128 and UL130, crucial for viral entry into non-fibroblast cells. Edited viruses showed significantly reduced infectivity in epithelial cells, confirming functional disruption of the PC. Plaque purification yielded isogenic clones with phenotypes comparable to AD169, a naturally PC-deficient strain. Furthermore, multiplexed editing created precise 663 bp deletions in over 60% of viral genomes. Importantly, this method enables HCMV editing in physiologically relevant cell types without fibroblast passaging, which typically introduces mutations. This SeV-Cas9 system represents a significant advancement for studying HCMV biology in diverse cell types.