Cas-CLOVER-mediated knockout of STAT1: A novel approach to engineer
packaging HEK-293 cell lines used for rAAV production
Abstract
In addressing the limitations of CRISPR-Cas9, including off-target
effects and high licensing fees for commercial use, Cas-CLOVER, a
dimeric gene editing tool activated by two guide RNAs, was recently
developed. This study focused on implementing and evaluating Cas-CLOVER
in HEK-293 cells used for rAAV production by targeting the STAT1 locus,
which is crucial for cell growth regulation and might influence rAAV
production yields. Cas-CLOVER demonstrated impressive efficiency in gene
editing, achieving over 90% knockout success. Selected HEK-293 STAT1 KO
sub-clones were subjected to extensive analytical characterization to
assess their genomic stability, crucial for maintaining cell integrity
and functionality. Additionally, rAAV9 productivity, Rep protein pattern
profile and potency, among others, were assessed. Our study also
established a comprehensive analytical workflow to detect and evaluate
the gene knockouts generated by this innovative tool, providing a solid
groundwork for future research in precise gene editing technologies.