The CRISPR‒Cas9 system for genome editing of the ASS1 gene in human cells to predict its effect on HSV-1 replication

Herpes simplex virus type 1 (HSV-1) is a highly contagious pathogen that establishes lifelong latent infections. The replication of HSV-1 is potentially influenced by the arginine succinate synthase (ASS1) gene, a key regulator of cellular metabolism. This study utilized the CRISPR–Cas9 genome editing platform to specifically target and disrupt the ASS1 gene to examine its effect on viral propagation. A guide RNA (gRNA) was designed to complement a sequence within the ASS1 gene. A donor plasmid and the pCas-guide plasmid were cloned and cotransfected into the human embryonic kidney (HEK) cells with sheared adenovirus (Ad)5 DNA (HEK293-AD) cells. Potential ASS1-knockout clones were identified and validated via polymerase chain reaction (PCR) and DNA sequencing analysis. The impact on HSV-1 replication was quantified via a plaque assay to determine the viral titer. Sequencing data from ASS1-gRNA/Cas9-treated cells did not confirm successful gene knockout, as the intended ASS1 disruption was not achieved. The viral titer did not significantly differ between the HSV-1 infection group (MOI=0.01) and the control group. These findings indicate that the single gRNA designed for this study lacked sufficient specificity to elicit a CRISPR-Cas9-mediated gene knockout. Therefore, employing a set of more specific gRNAs is recommended to increase targeting efficiency. Further investigations are needed to elucidate the desired genetic modification and observe its subsequent effects on HSV-1.