Gene Editing Technology: CRISPR-based Cas-CLOVER

Efficiency

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

Accessibility

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

Cas-CLOVER vs CRISPR/Cas9: Mechanisms and Advantages

CRISPR/Cas9

CRISPR/Cas9 uses a single guide RNA(gRNA) and the Cas9 nuclease to bind and cut DNA, enabling targeted genome edits through blunt-ended double-strand breaks. Repairs occur mainly through Non-Homologous End Joining (NHEJ), creating insertions or deletions at the cut site.

Because Cas9 is monomeric and relies on a single gRNA, the system is known to generate unintended off-target edits and genomic rearrangements. In addition, CRISPR/Cas9 lacks a clear freedom-to operate (FTO) landscape for many commercial applications.

Cas-CLOVER

Cas-CLOVER is a gene-editing system that uses two paired gRNAs and a dimeric architecture to improve precision and reduce off-target activity relative to CRISPR/Cas9. DNA binding is mediated by a deactivated Cas9 (dCas9), while cleavage occurs only when the Clo051 nuclease domains dimerize.

This configuration produces larger 4-bp overhang deletions, supporting more complete knockouts and more efficient knock-ins. Cas-CLOVER also occupies its own intellectual property space, providing freedom to operate for commercial cell line development and bioprocessing applications.

Feature / Mechanism	CRISPR/Cas9	Cas-CLOVER
Cutting Architecture	Single nuclease, monomeric	Dimeric system requiring two gRNAs
Guide RNAs	One gRNA	Two gRNAs for coordinated targeting
Type of DNA Break	Blunt-ended cut	4-bp staggered overhang
Off-Target Profile	Higher off-target risk; reported genomic rearrangements	Reduced off-target events due to dual-gRNA requirement
Repair Outcomes	Indels through NHEJ	Larger, cleaner deletions and more complete knockouts
Knock-In Precision	Variable efficiency	More efficient & predictable due to staggered overhangs
FTO / IP Position	Restricted by multiple patent estates	Independent IP position with clearer FTO
Use Case Fit	Basic gene editing and initial discovery work	High-precision editing, cell-line development, and commercial applications

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

Explore Licensing

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.

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.