CRISPR/Cas9 has demonstrated high efficiency gene editing in non-commercial settings, however, there are still questions about the commercial freedom to operate of the system, leaving many potential users on the sidelines. In addition, CRISPR/Cas9 uses a single guide RNA which has been shown to create “off targets” mutations or genomic cuts in unwanted sites. Hera’s alternative high-precision dimeric Cas-CLOVER gene editing technology on the other hand is supported by clean issued patents and has undetectable off-target activity. The Cas-CLOVER system maintains the high efficiency (1), and ease of use advantages of CRISPR/Cas9. Hera BioLabs offers commercial and research licenses as well as proof of concept services for this technology in drug discovery & early development.

Cas-CLOVER

  • Exclusively licensed technology and sub- licensable by Hera
  • Functionally similar to other CRISPR/Cas9 technologies, but uses a different nuclease protein called Clo51, which is covered under a set of patents distinct from other  CRISPR/Cas9 technologies
  • Cas-CLOVER is a fusion protein that comprises a nuclease-inactivated Cas9 protein fused to the Clo51 endonuclease
  • Cas-CLOVER achieves greater specificity through utilization of two guide RNAs as well as a nuclease activity that requires dimerization of subunits associated with each guide RNA

CHO knockout case study for bioprocessing applications

Chinese hamster ovary (CHO) cell expression systems are ideal for bioprocessing human and animal health therapeutics. During cell line development, selecting for high expressing clones has been a past challenge.  Glutamine Synthetase (GS) expression is essential for CHO cells viability. Reducing its expression via knockout results in cell death unless replaced by exogenous GS from media or stably integrated cassette. The expression of a stably integrated GS gene is proportional to the expression of a protein of interest (for example a monoclonal antibody) and this system is extremely effective for rapidly selecting high expressing pools or clones. Our successful protocol optimization and gene knockout in CHO is a demonstration of Hera’s ability to optimize the Cas-CLOVER system for your cell line of interest.
GS null CHO cells have traditionally been accessible through cost prohibitive licensing terms that may require high upfront fees, milestones and royalties. Hera and its partner Demeetra AgBio are providing single upfront fee license structure for Cas-CLOVER and GS knockouts for commercial bioprocessing and research.

20% – 40% cutting efficiency with Cas-CLOVER achieved

Cutting efficiency can be increased by repeating rounds of transfection (tf-rd1 & 2)

CAS-CLOVER activity is comparable to Cas9

  • Side-by-side comparison of CRISPR/Cas9 or Cas- CLOVER disruption of surface TCR expression in resting human T cells as assessed by FACS. Single or multiplexed (left (L) + right (R)) TCRα-specific gRNAs were tested
  • In contrast to CRISPR/Cas9, Cas-CLOVER requires both paired L and R gRNAs for on-site targeting
  • Additional targets were tested (β2M) and data is summarized by bar graph
  • Cas-CLOVER demonstrated comparable efficiency to CRISPR/Cas9 in disrupting the target gene
  • Cas-CLOVER’s requirement of two guide RNAs with activity dependent upon relatively strict spacer lengths results in a highly specific genome editing tool.

Cas-CLOVER showed no off-target detected by NGS

PD1 target knockout by Cas-CLOVER system were conducted in resting T cells. Resting T cells without CasCLOVER (3 replicates) were used as a control. Genomic DNA was collected and the top predicted off-target sites were selected for target amplicon amplification and sequencing by NGS. No off-target mutations above background were observed by NGS sequencing, with most of the sites at 30,000 –100,000X coverage. Possible combinations of gRNAs with Cas-CLOVER are shown below.

  1. Xianghong Li et al. Cas-CLOVER™: A High-Fidelity Genome Editing System for Safe and Efficient Modification of Cells for Immunotherapy. 2018 Precision CRISPR Congress Poster Presentation, Boston, MA