The Clean Alternative
Demeetra is introducing the Cas-CLOVER technology for commercial bioprocessing as well as newly edited GS knockout CHO cell lines. Cas-CLOVER differs from CRISPR/Cas9 in that it is a dimeric nuclease system lacking detectable off-target mutagenesis. The speciﬁcity of Cas-CLOVER enables multiple rounds of targeting at one locus to increase indel frequency without introducing the risk of unwanted off-target mutations.
GS CHO knockout: Cas-CLOVER Validation
Glutamine Synthetase (GS) expression is essential for CHO cell viability. Knockouts for GS result in cell death unless replaced by an exogenous stably integrated GS gene that is proportional to the expression of a protein of interest (for example, a monoclonal antibody), and this system is exceptionally useful in rapidly selecting high expressing pools or clones.
Shown here are indel frequencies of 18% and 43% at the CHO GS locus for one and two rounds of targeting, respectively. These on-target frequencies in CHO cells are higher than reported for ZFN and comparable to those of CRISPR/Cas9.
Simple and Accessible Licenses
GS null CHO cells and gene editing technologies have traditionally been accessible only through cost-prohibitive licensing terms that may require high upfront fees, milestones, and royalties. Demeetra is providing simple licenses structures with accessible economic terms for Cas-CLOVER and GS CHO knockouts for commercial bioprocessing as well as proof-of-concept services.
CRISPR/Cas9 vs. Cas-CLOVER
Compared with CRISPR/Cas9, Cas-CLOVER offers:
PiggyBac Outperforms Leap-in Transposon
No significant differences were observed in growth and viability for piggyBac vs. control CHO cells (above), while productivity improvements are maintained (below) (3)