Effective Gene Editing for Commercial Use with Cas-CLOVER

Simple commercial licenses and industry-leading CRISPR FTO meets efficiency, precision and outstanding know-how transfer.

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Efficiency

Generate targeted knockouts and knock-ins with equal or greater efficiency compared to CRISPR/Cas9

Accessible

A single license with Demeetra offers industry-leading Freedom to Operate (FTO) and favorable terms. Streamline your CRISPR due diligence process and proceed efficiently

Knowledge Transfer

Our gene editing experts internally validate processes and subsequently transfer this knowledge to you, ensuring seamless adoption

CRISPR/Cas9

Previous technologies such as zinc finger nucleases (ZFNs) and TALENs required intense protein engineering. CRISPR/Cas9 on the other hand uses a single easily designed and synthetized guide RNA (gRNA) to direct the Cas9 nuclease to a target DNA sequence for binding and cutting, enabling targeted genome modifications. The system results in small, blunt-ended cuts and leverages cellular DNA repair mechanisms—Non-Homologous End Joining (NHEJ) or Homology-Directed Repair (HDR)—to introduce deletions or insertions (Indels) at the cleavage site. Since Cas9 is monomeric and a single guide RNA is used, CRISPR/Cas9 has been reported to have significant off-target mutations and creates genomic translocations.

A significant challenge to the commercial use of CRISPR Cas9 is the lack of clear Freedom to Operate (FTO).

Cas-CLOVER

Cas-CLOVER is a gene editing system that shares many benefits with CRISPR/Cas9 but has distinct advantages due to its unique components. Unlike CRISPR/Cas9, which uses a single guide RNA (gRNA) and the Cas9 enzyme to cut DNA, Cas-CLOVER employs two separate gRNAs and a deactivated Cas9 (dCas9) for DNA binding without cutting. Instead of Cas9, the cutting action is performed by the dimeric nuclease Clo051, enhancing the system's precision and significantly reducing unwanted and off-target mutations. Additionally, Cas-CLOVER creates larger 4-base pair overhang deletions, which can lead to more complete gene knockouts and improved efficiency for gene insertions (knock-ins).

The distinctive mechanics of Cas-CLOVER not only improve its editing fidelity but also establishes its own intellectual property space, providing freedom to operate independently from CRISPR/Cas9 patents.

Demonstrated Efficiency in Key Commercial Systems

Cas-CLOVER generates targeted knockouts and knock-ins with high efficiency. We have demonstrated this across various systems in-house. By carefully crafting protocols, we ensure our clients receive the necessary knowledge to commence their projects promptly and effectively.

Cell lines
Greater than 90% editing efficiency in CHO-K1 cells
Plants
Successful targeting of albino phenotype gene in plants
Yeast & Microbes
Red colonies demonstrate high efficiency knock-ins

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Larger deletions & improved knock-in efficiency

Clo051's nuclease activity results in staggered cuts leading to large deletions and improved knock-ins. Deletions ranging from 8 to 50 base pairs or more have been shown to cause complete disruption in gene expression. Publications on CRISPR/Cas9 indicate a knock-in limit of 7-10 kilobases, while Cas-CLOVER is capable of efficiently introducing at least 20 kilobases at a selected genomic site.

Editing efficiency in HEK293 cells was 92.3%, with larger deletions causing full knockout.

Exceptional Precision

Cas-CLOVER requires Clo051 dimerization for its activity, leading to minimal off-target effects. In peer-reviewed publications, Cas-CLOVER exhibits a maximum potential off-target indel rate of less than 1%, whereas CRISPR/Cas9 can reach up to 10%.

Publications

Validated In Mammalian Cells, Yeast, and Plants

Our expertise and field of use spans multiple industries, allowing for the integration of Cas-CLOVER and transposase technologies into a wide range of commercial applications.