2020-07-13| Technology

Cas – CLOVER: A New Dawn in Genome Editing Technology

by Tulip Chakraborty
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By T. Chakraborty, Ph.D.

In the last few decades, the rapid development of genome editing has transformed research on the human genome, enabling scientists to comprehend better, the role of a single-gene product in causing disease in an organism. In simple terms, gene editing can be defined as a process where an organism’s DNA can be altered by the addition or removal of genetic materials.

Genome editing technologies find uses in fields ranging from neurodegenerative diseases, metabolic diseases, oncology to hematological diseases and can be achieved in vitro and in vivo where the technology helps correct the gene and is also responsible for other targeted modifications.

The three leading genome editing platforms currently used by researchers are Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9). Both ZFN and TALEN require restructured enzymes to fit within the target sequence and need to be separately made for each case. Additionally, both technologies are extremely labor-intensive and expensive.



The CRISPR-Cas9 gene-editing technology has come to the forefront in recent times due to its simplicity compared to its peers. The nuclease protein Cas9 can be modified to recognize new binding sites by manipulating the guide RNA sequences. Furthermore, CRISPR-Cas9 has the potential to edit multiple locations simultaneously, making this technology easier, efficient, scalable, and an indispensable tool in biological systems research. Currently, scientists and researchers have gone a step further and modified the existing CRISPR-Cas9 technology to provide further targeted therapy. Cas-CLOVER (Clo51-dCas9 genome editing technology) is one such technology developed by Hera BioLabs based in Kentucky, USA.


Cas-CLOVER, a Cleaner Gene Editing Tool

The CRISPR-Cas9 gene-editing system is dependent on a single guide RNA, thus resulting in high off-target mutation rates. Due to these mutations, successive rounds of CRISPR-Cas9 meditated editing in a cell may give rise to complications concerning disease therapy. In contrast, the Cas-CLOVER system utilizes two separate guide RNAs to target the enzyme to the site of action specifically. It consists of catalytically inactive Cas9 (dCas9) fused to a Type IIS restriction endonuclease, Clo-51.

Clo-51 activity is dependent on the dimerization of the enzyme, thus limiting the off-target effect of CRISPR-Cas9. This system combines the best of both CRISPR-Cas9 and TALEN’s technologies. It has the efficiency of the CRISPR-Cas9 system (as it utilizes the guide RNA and the inactive Cas9) while having the specificity of the TALEN system by using two guide RNAs. Cas-CLOVER works best when the gap between the two guide RNAs is between 16-30 nucleotides.

Initial validation studies, as conducted by Hera BioLabs performed in T cells, showed that the Cas-CLOVER system with two guide RNAs had similar efficiency as the CRISPR-Cas9 system. On the contrary, using only one guide RNA did not affect the target gene, validating that this technology only works once the Clo-51 enzyme dimerizes. Furthermore, this technology has also been used in plants like tobacco, and an experimental system like yeast suggesting the vast potential of this new technology [1].


Generation of Allogeneic Off the Shelf CAR-T Cells

Chimeric Antigen Receptor-T cell (CAR-T) therapy is an immunotherapy advance that has mostly been used in hematological tumors with modest success in solid tumors. In this therapy, the host immune system is manipulated to enhance its response to fight and eradicate the cancer cells. Allogeneic CAR-T cells are generally derived from a healthy patient. They are genetically manipulated to express certain receptors that will be able to mount an immune response, and then be introduced in the cancer patients. The advantage of this type of CAR-T cells over autologous cells derived from the patient is that it can be off the shelf and hence faster, cheaper, and easily accessible.

On the other hand, allogeneic T-cells may mount a graft vs. host response in the patient, which may be deadly. Further, during multiple rounds of genetic engineering, most of the cells reach a mature T-cell stage, which will not be able to mount the desired immune response. To avoid these problems and increase the population of T-memory stem cell (Tscm), that persists in the patient, Poseida Therapeutics, the sister company of Hera BioLabs, utilized the Cas-CLOVER system to develop an allogeneic CAR-T cell therapy, P-BCMA-ALLO1.

To avoid lethal side effects, the T-cell receptor (TCR) has to be knocked out completely using Cas-CLOVER technology while simultaneously knocking out MHC-I to increase the persistence of these cells and increase the population of Tscm. Due to the specificity and efficiency of this newly developed system, no known off-target effects were observed. Target cell killing and cytokine assay further showed that the allogeneic, genetically altered T-cell is functionally comparable to the non-edited T-cells, further confirming the technique’s efficiency. This new technology helped develop a fully allogeneic CAR-T cell product candidate with similar tolerability and safety profile as an autologous CAR-T product P-BCMA-101 [1,2].


Future Directions

Gene-editing technology has found innumerable uses in the development of cell imaging, gene expression regulation, therapeutic drug development, functional gene screening, and gene diagnosis, among others. The off-target effect in the implementation of CRISPR-Cas9 gene-editing technology has been optimized by the revolutionary Cas-CLOVER technology [3]. With further improvements and global collaboration amongst the world’s ever-growing research community, it is reasonable to assume that genome editing technology can uncover biological mechanisms underlying many diseases and provide novel therapies to facilitate life science development [4].


Hera BioLabs

Hera BioLabs is a technology provider with products and services surrounding their proprietary gene-editing technologies, including the piggyBac transposase system, the Cas-COLVER, and the TAL-CLOVER gene-editing system. The technologies are made available under a research license. The company also uses these techniques to provide contract research services and offers custom gene-editing services for the development of disease models.

Editor: Rajaneesh K. Gopinath, Ph.D.

Related Article: Proximity RNA Sequencing: Breaking New Grounds in Spatial Transcriptomics



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