CRISPR/Cas9 has become the benchmark for gene editing in non-commercial applications. However, its limited commercial freedom to operate and reliance on a single guide RNA often result in off-target mutations and low knock-in efficiency, presenting challenges for commercial and GMP bioprocessing use.
Demeetra’s high-precision dimeric Cas-CLOVER gene editing technology offers robust commercial freedom to operate and is available for commercial cell line development (CLD) and bioprocessing applications.
This proven alternative to CRISPR/Cas9 was recently validated in CHO cells and maintains the high efficiency and ease of use associated with CRISPR-based tools. Hera BioLabs, a fully owned subsidiary of Demeetra, holds a commercial license to Cas-CLOVER and leverages this platform to engineer improved CHO host cell lines and enable targeted knock-ins as part of our cell line development services.
CRISPR/Cas9
- Earlier technologies like ZFNs and TALENs required complex protein engineering.
- CRISPR/Cas9 introduced a simpler approach using a single, easily produced guide RNA (gRNA) to direct the Cas9 nuclease.
- Cas9 produces blunt-ended cuts with high efficiency, but this often leads to reduced knock-in success and an increased risk of unintended chromosomal alterations.
- Due to its single-guide design and monomeric nature, CRISPR/Cas9 is prone to significant off-target mutations.
- A major commercial limitation of CRISPR/Cas9 is the lack of clear Freedom to Operate (FTO).
Cas-CLOVER
- Shares many functional benefits with CRISPR/Cas9 but with important enhancements.
- Utilizes two guide RNAs and a deactivated Cas9 (dCas9), instead of relying on a single guide RNA.
- DNA cleavage is performed by a dimeric nuclease, Clo051, instead of Cas9, improving specificity and reducing off-target effects.
- Produces larger 4-base pair overhang cuts, enabling comparable knockout efficiency and improved knock-in efficiency.
- Novel intellectual property space, along with comprehensive gRNA and dCas9 licenses offers commercial Freedom to Operate (FTO) for CLD and bioprocessing.
Case Study: Development of CleanCut GS CHO Cells Using Cas-CLOVER
Overview
Hera BioLabs, a Demeetra company, applied the Cas-CLOVER gene editing system to develop a next-generation CHO host cell line—CleanCut GS—optimized for biomanufacturing. This platform enabled a precise knockout of the glutamine synthetase (GS) gene in a high-performance CHO suspension background, supporting robust selection of high-producing clones.
Hera first adapted adherent CHO-K1 cells to suspension growth in animal-free media. Cas-CLOVER guide RNA pairs were then designed for targeted knockout of the GS gene, and cells were transfected using the Nucleofector system. Single-cell clones were subsequently screened for both transient and stable expression performance, with evaluations conducted in-house and by external partners.
Sensitivity of CleanCut GS CHO clones to glutamine.
Cell lines show consistently decreased viability in medium without glutamine, indicating a full knockout phenotype.
Results
- Fully validated GS knockout confirmed by genomic sequencing.
- Multiple clones exhibited glutamine sensitivity, confirming successful GS gene knockout and the expected selection phenotype.
- Compatible with Harbor-IN transposase for stable gene insertion and scalable bioproduction.
- Stable CleanCut GS cells expressing Trastuzumab, a monoclonal antibody (mAb), have achieved high titers and industry-leading cell-specific productivity, demonstrating the platform’s suitability for biotherapeutic manufacturing.
- Freedom to operate—CleanCut GS is available for commercial use with no downstream IP royalties.
Conclusion
By integrating Cas-CLOVER’s high-efficiency editing with proprietary suspension CHO cells, Hera BioLabs developed a royalty-free CHO platform optimized for supporting high-yield biotherapeutic production through its cell line development services.
Cas-CLOVER and the CleanCut GS cell line are available for commercial license from our Parent Company, Demeetra.