Published in Biotechnology Journal

Abstract

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.

In summary, this study demonstrates the applicability of the Cas-CLOVER system in an rAAV-producing HEK-293 cell line. The high editing efficiency and minimal off-target effects make Cas-CLOVER a promising alternative to the standard CRISPR/Cas9 system. Although single STAT1 knockouts did not enhance rAAV yield, the potential for engineering cell lines for improved AAV production remains substantial. Leveraging gene-editing tools like Cas-CLOVER, alongside the genetic diversity of HEK-293 cells and high-throughput technologies, could lead to significant yield improvements.

Peter Andorfer, Carolin-Isabel Kahlig, Doris Pakusic, et al. Cas-CLOVER-mediated knockout of STAT1: A novel approach to engineer packaging HEK-293 cell lines used for rAAV production. Biotechnology Journal.