Though CRISPR-Cas enabled targeted genome engineering across a vast array of organisms, the system features major disadvantages. Frequent off-target mutagenesis, licensing restrictions and non-ideal economic license terms have inhibited commercial crop-science product upscaling, and, in many cases, entirely disqualified CRISPR’s use by many commercial crop developers.
Seeking an alternative to the single-guided Cas9 editing system for crop scientists, Demeetra AgBio set out to validate its proprietary Cas-CLOVER technology by confirming the activity and cutting efficiency of Cas-CLOVER in tobacco.
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How Cas-CLOVER Works And How It Differs From Single-Guided Cas Gene Editing
Cas-CLOVER is a patented dimeric gene editing system using a pair of guide RNAs (gRNAs), nuclease-inactivated proteins fused to our proprietary Clo051 endonuclease.
Figure 1: Our Cas-CLOVER gene editing system. Note the presence of left gRNA, right gRNA, and the two (2) Clo51 subunits
The fusion protein serves only as a linker between the guide pairs and Clo51; it is mutated and unable to cut DNA. Cleavage activity thus depends on the dimerization of the “obligate dimer” Clo51.
How Researchers Demonstrated Optimized Cas-CLOVER Cutting Efficiency In Plants
Cas-CLOVER has demonstrated robust targeted mutagenesis in mammalian cells, including human T-cells, iPSCs and chinese hamster ovary (CHO) cells, but we wanted to open the door to Cas-CLOVER use in plants, trait discovery, and crop science applications.
Our strategy involved targeting the phytoene desaturase (PDS) gene in tobacco due to its visually pale and white phenotype. We focused the screening at the sequence level on two guide pair sites called 1 and 4. Green, pale mixed, and white shoots were all screened to determine insertion and deletion “indel” efficiencies, a common measurement for genome editing. Then, 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).
Figure 2: Shoot phenotypes. Note the contrast between wild-type (green) from successfully manipulated PDS gene phenotype (white)
The full white phenotype shown here indicates all 4 alleles were knocked out with Cas-CLOVER. After fully optimizing our protocols we enhanced both the number of white/pale shoots per plate and genomic cutting efficiencies up to 90%+ as shown below.
Figure 3: Phenotype of successfully edited tobacco shoots
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 shown below as indel percentages.
Figure 4: Estimated indel frequencies of individual plant subjects
Improved Downstream Processes For Cas-CLOVER Editing Through The plant T1 Generation
Eight (8) T0 edited plants were grown to the flowering stage, the plants were self-pollinated, and then the seeds were harvested. Following seed germination, seedlings were transferred to magenta boxes and grown large enough to check for edit stability in this T1 generation. Unpurified PCR products were screened using an optimum resolution on an automated DNA analysis machine, QIAxcel (<500bp size).
- Cas-CLOVER produces larger indels than other technologies
- The large indels are rapidly and easily detected by peak size & translated into a gel image
Figure 5: QIAxcel DNA analysis machine
Figure 6: QIAxcel results demonstrating very high stability in the edited genome.
Figure 7: QIAxcel results, showing deletion versus wild type reads
T1 indels were then double checked using Synthego ICE. 14 of 16 samples sequenced and analyzed demonstrated 100% indel percentage. Two samples showed indel and WT sequences by both QIAxcel and ICE. Two other samples with WT peaks by QIAxcel revealed edited but smaller deletions by ICE not picked up in the QIAxcel screen.
Figure 8: Synthego ICE results reflecting 100% indel for 14 of the 16 specimens and partial indel for the other 2
Demeetra’s Approach To Cas-CLOVER Licensing
Overall, we concluded that Cas-CLOVER performed optimally as an alternative to existing technologies for crop science. The highly efficient two-guide Cas-CLOVER technology also offers commercial freedom-to-operate (FTO) in a simple single license and flexibility for AgTech companies. With Cas-CLOVER activity validated in plants, we are excited to help scientists in crop trait discovery and development harvest unlimited opportunities.
Cas-CLOVER uses Clo51, a nuclease covered under a set of issued patents distinct from the labyrinthine CRISPR patents. There are no known legal entanglements with our Cas-CLOVER patents. Demeetra AgBio holds the exclusive licenses in Agriculture and we offer sublicenses and strategic development partnerships as follows.
- Evaluation License: Technology transfer with reagents and protocols for a set evaluation period. No risk, you just pay for the low cost of the reagents. Check out our online shop.
- Research License: Economically attractive yearly fee. Freedom to switch technologies for commercialization to navigate gene editing regulatory landscape
- Commercial License: Flexibility of one-time fee-based license or economical standard upfront/milestone/royalty structure
Learn more about our research and how our team can help move your studies forward by scheduling a call with us.