In spite of a plethora of mammalian cell expression systems available for use in therapeutic biomanufacturing, the production of biotherapeutic proteins, antibodies, viral vectors and vaccines remains time-consuming and expensive. Contributing to this costliness are inefficiencies in the platform cell lines (i.e. CHO and HEK293 cells).
Targeted gene editing technology Cas-CLOVER is effective in all cell types and can be applied to myriad projects like targeted knock-ins, broad platform improvements like glutamine synthetase (GS) high-yield clonal selection strategies, and more.
The Cas-CLOVER system is a hybrid gene editing technology using nuclease-inactivated Cas protein fused to Clo051endouclease. DNA cleavage activity depends on the dimerization of an “obligate dimer” of Clo051 (Figure 1) resulting in targeted knockout or knock-in through non-homologous end-joining (NHEJ) or homologous directed repair (HDR) with more on-target fidelity than other gene editing technologies.
With Cas-CLOVER, researchers can thus access unlimited potential for cell line and biotherapeutic improvement.
Figure 1: Architecture of Cas-CLOVER. Two gRNAs form a pair and bind to DNA through fusion protein dCas. The spacer between the gRNAs is flexible at 16-30 bp enabling accessible target design at any locus. The dCas is fully inactivated and does not cut the DNA, eliminating off-target problems with CRISPR/Cas9, but instead the obligate dimer nuclease Clo051 cuts the DNA specifically when guided to the on-target site.
The Power of Clean, Targeted Knockouts
Using Cas-CLOVER to knockout gene(s) can result in vast improvements to bioprocessing, and in the case of glycoengineering, this method can produce entirely new and improved products.
Some gene targets are project-specific; pesky proteins that come off the purification column at the same time as the target molecule can be removed from the equation upstream by gene knockout.
Other modifications have broad applications for many projects. In CHO cells when the endogenous glutamine synthetase (GS) gene is knocked out and restored by integration along with the gene of interest (GOI), a six-fold increase in high-producing cell clones can be achieved.
We leveraged Cas-CLOVER’s flexible spacer region in dual-guide design with 17 gRNA pairs targeting glutamine synthetase (GS) in suspension CHO cells that targeted Exon 1 (pairs 1-4) or Exon 5 (pairs 5-17). Overall editing efficiencies in cell pools ranged from 10-40% in a single transfection step (Figure 2), which enabled bountiful GS knockout single-cell clones.
Figure 2. Glutamate synthetase (GS) knock-out in suspension CHO cells. Expected length of WT Exon 1 is 876 bp and WT Exon 5 is 752 bp. A) Lanes 1-4 are guide RNA pairs that target Exon 1 followed by the WT CHO. Lanes 5 and 6 are guide RNA pairs that target Exon 5 again followed by WT. B) Lanes 1-9 are guide RNA pairs that target Exon 5. The final lane 10 is WT CHO.
Knock-In Using Cas-CLOVER
Unstable and variable transgene expression associated with random integration of a plasmid has long plagued cell line development (CLD) efficiencies. These issues often delay projects and cause lag in the cell line development pipeline. To remedy this common issue in upstream processing, Cas-CLOVER can be used for efficient site-specific integration of one or multiple copies of the expression gene.
The expression cassette can be targeted to well-defined loci in mammalian cells, thus delivering high-yield homogenous protein production. In our proof-of-concept work the Hipp11 (H11) gene was targeted as it is well-characterized as a “safe harbor” locus for gene knock-in within CHO-S cells.
Our results demonstrate that, by using the Cas-CLOVER system, CLD teams can achieve targeted knock-ins in as little as two weeks. Following a short 10-17 day cell culture and selection process, the knock-in efficiency was an estimated 80-90%. Targeted integration of a 4kb donor vector at the H11 safe harbor site was confirmed by sequencing as shown below.
Figure 3. Targeted knock-in at the H11 site was confirmed by TOPO cloning and sequence analysis. Both the 5’ and 3’ junctions between the arms of homology of the donor vector and the H11 target genomic sequences were confirmed and highlighted in blue. The donor vector contained piggyBac inverted terminal repeats (ITRs) which can be used for Footprint-Free removal of selection cassettes. piggyBac ITRs require a “TTAA” site for excision, this is highlighted in red above.
Cas-CLOVER Bioprocessing Evaluation and Commercial License Opportunity
Demeetra has exclusive rights to gene editing technology Cas-CLOVER for pharmaceutical bioprocessing with commercial freedom to operate (FTO). With a risk-free Cas-CLOVER reagent purchase at our shop and evaluation license, our users receive:
- Access to technology being used by Big Pharma, CDMO’s and Biotech alike
- Validated Cas-CLOVER gRNA pair target site design at important knockout and knock-in loci
- Supply of Cas-CLOVER mRNA
- Protocols and tech support including for suspension CHO cell editing
Following successful evaluation, we offer highly accessible (a fraction of CRISPR/Cas9, with non-royalties options) commercial licenses! Licensing, procurement, and business development teams can also contact us for more information on licensing.
References
- Zhu F, Gamboa M, Farruggio AP, Hippenmeyer S, Tasic B, Schu ̈le B, et al. DICE, an efficient system for iterative genomic editing in human pluripotent stem cells. Nucleic Acids Res. 2014; 42.
- Simon H, Youn YH, Moon HM, Miyamichi K, Zong H, Wynshaw-Boris A, Luo L. Genetic Mosaic Dissection of Lis1 and Ndel1 in Neuronal Migration. Neuron. 2010; 68:694–709.
- Tasic B, Hippenmeyer S, Wang C, Gamboa M, Zong H, Chen-Tsai Y, et al. Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc Natl Acad Sci. 2011; 108:7902–7. https://doi.org/10.1073/pnas. 1019507108 PMID: 21464299.
- Chi X, Zheng Q, Jiang R, Chen-Tsai RY, Kong L-J (2019) A system for site-specific integration of transgenes in mammalian cells. PLoS ONE 14(7): e0219842. https://doi.org/10.1371/journal.pone.0219842.