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, recently validated in CHO cells, 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.

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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

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.

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