Cas-CLOVER for Creating Improved Platform Cell Lines and Animal Models

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The Cas-CLOVER™ Site-Specific Gene Editing System is being used to engineer platform models for basic research, drug discovery and development, bioproduction, and animal model creation. Cas-CLOVER is a proprietary dimeric nuclease system that utilizes Clo051 nuclease and forward and reverse gRNA pairs for highly specific on-target mutagenesis, thus minimizing off-target activity. The targeted nuclease activity results in modifications through the homology-directed repair (HDR) or the non-homologous end joining (NHEJ) mechanisms and can be designed to produce relatively large deletions to ensure either loss of gene function or targeted knock-in for protein expression.

Cell Line Development

As indications for biologic drugs continue to expand and the demand for biosimilars grows, cost effective improvements in bioproduction platforms become central to enhancing performance during development and manufacturing. Hera BioLabs has demonstrated Cas-CLOVER to be a high-efficiency targeted gene editing tool for use in HEK293 and CHO cells, base cell lines validated for bioproduction.

Hera has used Cas-CLOVER to create cell lines with highly sought modifications, such as fucosyltransferase 8 (FUT8) and glutamine synthetase (GS) knockouts (KO), for consistent and rapid identification of stable, high-expressing CHO cell clones. Efficient production of our base CHO cells lacking the GS gene is shown in Figure 1A.

Figure 1. Cas-CLOVER
Figure 1. (A) Cas-CLOVER shows 20–40%+ cutting efficiency at the glutamine synthetase (GS) comparable to the CRISPR-Cas9 activity with the same (L and R) guide RNAs. (B) Cas-CLOVER shows high efficiency (80–90%) targeted knock-in of protein expressing-genes into the H11 safe harbor loci.

In addition to using Cas-CLOVER to create the GS KO CHO cell line, Hera uses its proprietary technology for targeted knock-in of human genes to safe harbor loci of cell lines to provide stable and predictable overexpression of proteins of interest without negatively affecting cellular characteristics. For example, our scientists have developed Cas-CLOVER reagents and protocols for targeting at the H11 locus in CHO suspension cultures for extremely high (80–90%) target knock-in efficiency (Figure 1B). Our cell line modification methods are further streamlined using automated workflows for single-cell cloning with image-based documentation. Hera is using our developed Cas-CLOVER platforms for bioproduction services with clear freedom to operate.

Animal Model Creation 

Hera has been applying Cas-CLOVER to animal model creation to allow gene cutting across a flexible spacer region between paired gRNAs. This important attribute enables design of large on-target deletions (8–50 base pairs) to increase likelihood of gene function disruption and rapid screening of knockouts, attributes which contribute to accelerating timelines and improving project success.

Large deletions provide confidence in selecting founder transgenic animals for breeding and characterizing gene expression. For example, Hera has demonstrated large deletions using the Cas-CLOVER system to disrupt target rat genes. Of 26 rats produced from Cas-CLOVER-transfected embryos, 16 rats (61%) had at least one gene disruption in the region of interest with the average INDEL of 27 base pairs in length (range: 3 to 744 base pairs); see Figure 2.

Figure 2. Rat embryo electroporation with Cas-CLOVER
Figure 2. Rat embryo electroporation with Cas-CLOVER yields 16/26 F0 rats with INDELs ranging in size from 3 bp to 744 bp, including several animals with biallelic mutations.

Immunodeficient rodent models are a vital tool for oncology (xenograft/PDX), immuno-oncology, and infectious disease research. Hera has shown that Cas-CLOVER has high activity in targeting the Il2rg gene to create knock-outs in both mice and rats which contributes to a highly immunodeficient phenotype. Hera’s SRG rat™ (OncoRat®) harbors an Il2rg and Rag2 gene knockout leading to a highly immunodeficient phenotype.

Hera’s SRG rat is an excellent host for xenografts and PDXs on a robust Sprague–Dawley background ideal for drug or cell-based efficacy and/or toxicology applications. The SRG rat’s larger size allows for easier surgical manipulation enabling orthotopic or device implantation, serial blood/tumor tissue sample collection, etc. The SRG rat is being bred and distributed by Charles River Laboratories for research use.