Gene Editing in plants is stable, efficient, and reliable with Cas-CLOVER, the clean alternative to CRISPR/Cas9

Date:04-04-2022

Though CRISPR-Cas9 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.

How Cas-CLOVER works and why it differs from gene editing with single-guided Cas

Cas-CLOVER is a patented dimeric gene editing system that uses a pair of guide RNAs (gRNAs) with nuclease-inactivated proteins fused to our proprietary Clo051 endonuclease.

Figure 1: Our Cas-CLOVER gene editing system
Note presence of left gRNA, right gRNA, and the two Clo051 subunits

The fusion protein serves only as a linker between the guide pairs and Clo051; it is mutated and unable to cut DNA. Because of this, cleavage activity depends on dimerization of the “obligate dimer” Clo051.

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 Demeetra also wanted to open the door to Cas-CLOVER’s use in plants, trait discovery, and crop science applications.

Demeetra’s strategy involved targeting the phytoene desaturase (PDS) gene in tobacco, due to its visually pale and white phenotype. Demeetra’s researchers 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 in genome editing. The researchers then 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)
Figure 3: Successfully edited tobacco shoot phenotypes

The full white phenotype shown above indicates all 4 alleles were knocked out with Cas-CLOVER. After fully optimizing our protocols, the team 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, Demeetra’s researchers analyzed individual transgenic plants using a combination of PCR, Sanger sequencing, and applications like Synthego ICE, with indel percentages shown on the next page.

*Indel percentage estimated using three different programs performing decomposition analysis of PCR product Sanger sequencing results.
Figure 4: The estimated indel frequencies of individual plant subjects

Downstream processes for Cas-CLOVER editing improved with plant T1 generation

Eight T0 edited plants were grown to the flowering stage, self-pollinated, and then harvested for the seeds. 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 Cas9

• The large indels are rapidly and easily detected by peak size & translated into a gel image

Figure 5: QIAxcel DNA analysis machine

QIAxcel T1 Plant Knockout Gel

Figure 6: QIAxcel results demonstrates high stability in edited genome

QIAxcel Mutagenesis Report

Figure 7: QIAxcel results that show deletion versus wild type reads

T1 indels were then double checked using Synthego ICE. 14 out of 16 samples sequenced and analyzed demonstrated 100% indel formation. 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 that were 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

Read more on Demeetra AgBio's website.