Cas-CLOVER-mediated Knockout of STAT1: A Novel Approach to Engineer Packaging HEK-293 Cell Lines Used for rAAV Production

To address the limitations of CRISPR-Cas9, including off-target effects and high licensing costs for commercial use, the Cas-CLOVER system, a dimeric gene-editing tool activated by two guide RNAs, was recently developed. This study evaluates the application of Cas-CLOVER in suspension HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the STAT1 locus, a critical regulator of cell growth that may influence rAAV production yields.

Cas-CLOVER demonstrated high editing efficiency, with mutations detected in all sequencing reads for 12 of the 13 clones analyzed, representing a success rate of 92.3%, consistent with the highest values reported in the literature. All target loci were edited, with no wild-type sequences detected among the edited clones. The mutations primarily consisted of various deletions, with one instance of an insertion, ranging from 3 to 171 base pairs. These larger deletions increased the likelihood of achieving a disruptive knockout, as evidenced by the absence of detectable STAT1 protein via Western blot analysis, regardless of whether the genomic alterations resulted in a frameshift with a subsequent stop codon or an in-frame deletion.