CRISPR-Cas9 screening reveals TM9SF2 knockout as a solution to HEK293 cell aggregation for improved AAV production

The inherent tendency of human embryonic kidney (HEK) cells to aggregate in suspension culture poses a significant obstacle to the large-scale production of adeno-associated virus (AAV) vectors for gene therapy. To overcome this, we employed a virus-free CRISPR-Cas9 single gene knockout (KO) library in HEK293T cells to systematically identify key genetic regulators of cell aggregation. By serially passaging the KO library and selectively sub-culturing only the suspended (non-aggregated) cells, we enriched for genotypes that reduce aggregation. Next-generation sequencing identified TM9SF2 as a key target, and its KO reduced aggregation by 53% and 28% in HEK293T and HEK293 cells, respectively. Transcriptomic analysis of TM9SF2 KO cells showed enrichment in MAPK signaling, calcium signaling, and plasma membrane components, offering insights into the mechanisms of aggregation suppression. Importantly, TM9SF2 KO did not compromise the genome titer, full capsid ratio, or infectivity of AAV5, AAV8, and AAV9, representative serotypes for secreted AAVs, in both HEK293T and HEK293 cells. These findings identify TM9SF2 as a promising target to mitigate cell aggregation in HEK293-based systems, thereby facilitating scalable AAV vector production.