Increasing Cutting Efficiency in Plants with the Cas-CLOVER System

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Increasing Cutting Efficiency in Plants with the Cas-CLOVER System

The CRISPR-Cas9 system has been an effective technology for targeted genome engineering with the capability to efficiently edit a vast array of organisms. However, CRISPR-Cas9 also has a few major disadvantages, including off-target mutations and licensing restrictions. In recent years, these drawbacks have created multiple challenges with upscaling to a commercial product with this particular technology. 

As an alternative to the single-guided Cas9 system, Demeetra AgBio set out to validate our proprietary Cas-CLOVER technology by confirming the activity and cutting efficiency of Cas-CLOVER in the plant model organism tobacco.   

Cas-CLOVER confirmation of function in plants: Nicotiana tabacum RDR6 Knockout

Recently, we confirmed the activity of Cas-CLOVER in stably regenerated transgenic tobacco by targeted inactivation of the RDR6 gene, widely known to play a role in bioproduction of exogenous proteins. We analyzed the cutting efficiency of our Cas-CLOVER technology using four different pairs of guides targeting the RDR6 gene in tobacco. 

Results of initial proof-of-concept (POC) editing efficiencies in tobacco with Cas-CLOVER

The results we obtained from this initial study demonstrates that the Cas-CLOVER system exhibits a range of 10-30% performance in gene editing efficiencies, which is comparable to published results using CRISPR/Cas9. Regarding the potential for upscaling this product, we have found that RDR6 KO published with Cas-CLOVER in plants demonstrated POC in a potentially commercially valuable target. The chart below shows the distribution of cutting efficiencies in Cas-CLOVER edited transgenic plants by Synthego Ice and/or TIDE. 

Overall, we concluded that Cas-CLOVER was a comparable alternative to existing technologies, and believe that our proprietary two-guide Cas-CLOVER technology offers an effective alternative to Cas9 for gene editing, as well as having very little to no off-target mutations. However, comparable was not good enough for us, so we decided to optimize cutting efficiencies of Cas-CLOVER in tobacco. 

Optimizing the cutting efficiencies of Cas-CLOVER in tobacco

For our optimization experiment we targeted the phytoene desaturase (PDS) gene in Tobacco due to its visually pale and white phenotype. We  focused the screening at the sequence level on guide sites 1 and 4. Green, pale mixed, and white shoots were all screened to determine cutting efficiencies. Afterwards, we verified that only white and pale material was edited at an overall average of around 50%+ editing efficiency (white shoots/total shoots from plates).

After fully optimizing our protocols we enhanced both the number of white/pale shoots per plate and genomic cutting efficiencies up to 90%+. 

Genetic modification using Cas-CLOVER was first confirmed by Next-Gen sequencing. Additionally, we analyzed individual transgenic plants using a combination of PCR, sanger sequencing and applications like Synthego Ice and TIDE (Tracking of Indels by DEcomposition). 

Cas-CLOVER is Not Covered by CRISPR Patents

Cas-CLOVER uses Clo51, a nuclease covered under a set of patents distinct from the CRISPR patents. To our knowledge, there are no known legal entanglements with our Cas-CLOVER patents compared to other gene-editing technologies. Our technology doesn’t contain the ambiguity of CRISPR/Cas9 licensing. Now that we have validated genetic cutting in tobacco with Cas-CLOVER, we hope to be able to advance product development efforts in many other plant species around the world. 

Interested in learning more about Cas-CLOVER?

We hold the Cas-CLOVER licenses for research and commercial fields of therapeutic bioprocessing, industrial biotechnology & agriculture. We also offer licenses and strategic development partnerships with all of our products.

We offer clear commercial freedom to operate on any plant species, and simple accessible licenses to users. We feel the validated Cas-CLOVER activity in plants is exciting for crop trait development as it allows numerous opportunities. Learn more about our research and how our team can help move your studies forward by contacting us today!

References

Norman, D., Rector, K., Tateno, M., & Crawford, J. (2020). Targeted editing of Tobacco with Cas-CLOVER™: the clean alternative to CRISPR/Cas9 for plant genome editing. Plant Biology 2020. Demeetra AgBio, Lexington, KY. https://plantbiology2020.ipostersessions.com/Default.aspx?s=F7-2E-1F-D7-8F-B7-07-3F-C4-F2-8A-1C-5E-8B-08-F2 

 

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