Cas-CLOVER: The Clean Alternative to CRISPR/Cas9 For Bioprocessing

Why Cas-CLOVER Is Better Than CRISPR/Cas9

• Cas-CLOVER is a cleaner gene editing alternative to CRISPR/Cas9 for commercial bioprocessing similar indel frequencies can be achieved as compared with CRISPR
• Cas-CLOVER is a highly specific system demonstrating no detectable off-targets
• Cas-CLOVER (and TAL-CLOVER) are covered by issued patents with clear freedom-to-operate

Demeetra is offering evaluation licenses for Cas-CLOVER and GS CHO knockouts separately or together, which can convert into commercial licenses with very accessible terms, including single-time payments with no royalties. Special license structures are available for CDMOs.

Chinese Hamster Ovarian (CHO) cells are emerging as a robust platform for bioprocessing serving both early and late stage biotherapeutic drug supply. However, these cells, and other hosts (i.e. HEK293), can be optimized for even greater potential through advanced gene editing. For example, when the endogenous glutamine synthetase (GS) gene is knocked out in CHO cells, a 6-fold increase in high producing cell lines is achieved (1). In another study, CHO with ANXA2 and CTSD gene knockouts were introduced in order to eliminate CHO host cell proteins which complicate downstream processing and can contribute to immunogenicity (2).

Site-specific nucleases such as ZFNs, CRISPR and now Cas-CLOVER bind to the target sequence and generate insertions and deletions “indels” resulting in null mutations “knockouts” or exogenous “knock-ins” when supplied with a donor vector. If the indel frequency is determined to be high enough by sequencing, only a small set of cells need to be screened and cloned in order to identify edited cell lines, greatly increasing efficiency and decreasing timelines.

CRISPR/Cas9 technology has been utilized successfully in many cell lines. However, CRISPR suffers from two flaws, off-target mutagenesis, which we will discuss in more detail (Figures 3,4). In addition, intellectual property (IP) entanglements or lack of clear commercial freedom to operate have limited its use commercially.

Figure 1

Demeetra AgBio is a new biotech company stemming from the gene editing platforms of Transposagen which is best known for its commercialization of the piggyBac® transposon. piggyBac is now being utilized in drug discovery & development by Hera BioLabs, cell therapy clinical trials by Poseida Therapeutics and for human therapeutic cell biomanufacturing by Lonza.

Demeetra is introducing the Cas-CLOVER technology for commercial bioprocessing as well as newly edited GS knockout CHO cell lines. Cas-CLOVER differs from CRISPR/Cas9 in that it is a dimeric nuclease system lacking detectable off-target mutagenesis (Figure 5) and licensing is straight forward with issued IP and clear commercial freedom to operate.

Dimeric Cas-CLOVER uses two guide RNAs to target sequences of two “half-sites,” and creates indels by cutting at the spacer sequences between the two half-sites with the proprietary dimeric Clo51 nuclease. The key to specificity and ease of design for Cas-CLOVER is the relative stringency of the dimeric Clo51 nuclease, combined with a little wiggle room in the spacer region. Data was presented at a recent gene editing conference demonstrating that Cas-CLOVER is strictly controlled by on-site target binding of both gRNAs within a specific spacer length, ideally 16-30 bases (5). Yet that spacer length range enables enough flexibility to easily design Cas-CLOVER targets sites in most genes of interest.

Further, no single gRNA or mis-matched gRNA combinations which could lead to off-target mutations were observed by Next Generation Sequencing (NGS) in human T-cells edited with Cas-CLOVER. Most of the sites were analyzed with 30,000 –100,000X coverage. Possible combinations of mis-matched gRNAs with Cas-CLOVER are shown below the figure.

Figure 2

The specificity of Cas-CLOVER enables multiple rounds of targeting at one locus to increase indel frequency without introducing the risk of unwanted off-target mutations. Here we report indel frequencies ranging from 15% to 43% at the CHO GS locus for one and two rounds of targeting respectively. These on-target frequencies in CHO cells are higher than reported for ZFN and comparable to CRISPR/Cas9.

Figure 3

Below are examples of indels created by Cas-CLOVER when targeting the GS gene in CHO cells. The indels are within an exon of the GS gene and are expected to create a full knockout phenotype. Demeetra will be publishing a following up article on the characterization of the knockout clone CHO cell lines.

Figure 4

In contrast to Cas-CLOVER, the monomeric CRISPR/Cas9 nuclease system is directed to target sites with a single gRNA as depicted below.

Figure 5

Cas9 can still bind and cut even if there are multiple base-pair mismatches within the single gRNA, leaving the system at risk for significant off-target mutagenesis. In CHO cells, the single gRNAs that contained one or 2 base-pair mismatches resulted in 1-5% off-target indel rate. One of the Cas9 gRNAs resulted in a 10.7% off-target mutation rate; while 3 out of the 4 off-target sites screened for another gRNA contained off-target mutations.

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References

1. Fan et al. (2012) Improving the Efficiency of CHO Cell Line Generation Using Glutamine Synthetase Gene Knockout Cells. Biotechnology & Bioengineering
2. Fukuda et al. (2019) Anxa2- and Ctsd-knockout CHO cell lines to diminish the risk of contamination with host cell proteins. Biotechnology Progress.
3. Fu et al. (2013) High-frequency off-target mutagenesis induced by CRrIsPr-Cas nucleases in human cells. Nature Biotechnology
4. Lee et al (2015) Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway. Scientific Reports
5. Li et al (2018) Cas-CLOVER™: A High-Fidelity Genome Editing System for Safe and Efficient Modification of Cells for Immunotherapy. Precision CRISPR Congress Poster.