Cas-CLOVER nucleases – the clean alternative to CRISPR/Cas9

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Cas-CLOVER Gene Editing

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Hera BioLabs, now part of Demeetra, proudly offers Cas-CLOVER gene editing technology for commercial cell line development and bioprocessing applications.

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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 is extensively validated in both CHO and HEK293 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.

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CRISPR/Cas9

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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).

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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.

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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.[/fusion_text][fusion_text animation_direction=”left” animation_speed=”0.3″ animation_delay=”0″ hide_on_mobile=”small-visibility,medium-visibility,large-visibility” sticky_display=”normal,sticky” margin_top=”25″ margin_bottom=”0″ font_size=”18″]

Sensitivity of CleanCut GS CHO clones to glutamine.

[/fusion_text][fusion_imageframe custom_aspect_ratio=”100″ lightbox=”no” linktarget=”_self” align_medium=”none” align_small=”none” align=”none” hover_type=”none” magnify_duration=”120″ scroll_height=”100″ scroll_speed=”1″ caption_style=”off” caption_align_medium=”none” caption_align_small=”none” caption_align=”left” caption_title_tag=”2″ animation_direction=”left” animation_speed=”0.3″ animation_delay=”0″ hide_on_mobile=”small-visibility,medium-visibility,large-visibility” sticky_display=”normal,sticky” filter_hue=”0″ filter_saturation=”100″ filter_brightness=”100″ filter_contrast=”100″ filter_invert=”0″ filter_sepia=”0″ filter_opacity=”100″ filter_blur=”0″ filter_hue_hover=”0″ filter_saturation_hover=”100″ filter_brightness_hover=”100″ filter_contrast_hover=”100″ filter_invert_hover=”0″ filter_sepia_hover=”0″ filter_opacity_hover=”100″ filter_blur_hover=”0″ image_id=”9645|full” caption_text=”Cell lines show consistently decreased viability in medium without glutamine, indicating a full knockout phenotype.”]http://herabiolabs.com/wp-content/uploads/2025/08/GS-sensitivity.png[/fusion_imageframe][fusion_text animation_direction=”left” animation_speed=”0.3″ animation_delay=”0″ hide_on_mobile=”small-visibility,medium-visibility,large-visibility” sticky_display=”normal,sticky” margin_top=”5″]

Cell lines show consistently decreased viability in medium without glutamine, indicating a full knockout phenotype.

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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.

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Cas-CLOVER and the CleanCut GS cell line are available for commercial license from our Parent Company, Demeetra.

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