Like yeasts, E. coli and other microorganisms, plants have long been useful tools to produce recombinant proteins through genetic modifications. The goals of these modifications vary from producing therapeutic proteins to making proteins for industrial use. However, genetically altering organisms to produce a protein of interest is a time consuming and labor intensive process, but one well worth the effort. Researchers are constantly searching for ways to improve protein production yields and efficiency.
Roadblocks to High Protein Yields
Several endogenous plant proteins inhibit recombinant protein production in genetically modified plants. For example, the RNA silencing pathway, which enables plants to evade foreign agents such as viruses, isn’t able to distinguish the difference between deadly attackers and the viral vectors used to genetically alter the plant. An important player in the plant’s viral defense system is RNA-dependent RNA polymerase 6 (RDR6), which synthesizes dsRNA molecules that are eventually cleaved, resulting in small oligos that interfere or silence genes. Ultimately, this process hinders high levels of protein expression.
Due to RDR6’s involvement in the RNA silencing pathway, which lowers protein yield, Matsuo et al. utilized CRISPR gene editing technology to overcome this molecular hurdle. Matsuo and his team accomplished this by creating RDR6 knockout (KO) tobacco plants that reduced activity of the plant’s RNA interfering system, allowing for greater levels of protein production1. Figure 1 shows the difference in green fluorescent protein (GFP) protein expression between wild type (WT) and KO tobacco plants seen in the study.
Figure 1: GFP was transiently expressed using the 35S promoter of the caulifower mosaic virus (CaMV) in the ΔRDR6 and WT plants.
Cas-CLOVER Builds on the CRISPR-Cas Framework for More Precise Edits
Our company, Demeetra, validated the activity of Cas-CLOVER in plants by targeted inactivation of the RDR6 gene in tobacco. We achieved a knockout efficiency of at least 18% for our first target which is significant for tobacco. To learn more about this research, check out our poster presentation presented at this year’s virtual Plant Biology Worldwide Summit.
Edits such as knocking out the RDR6 gene may be valuable, as they can result in more robust recombinant protein production for rapid and scalable therapeutic manufacturing. In fact, the vaccine for the Ebola outbreak of 2014 was produced in transgenic tobacco, showing the value of these genetic editing tools in agriculture.
Novel gene editing proteins such as CRISPR-Cas9, transcription activator-like effector nucleases (TALEN), and zinc-finger nucleases (ZFN) target specific genes in a deliberate manner. This is compared to previous tools that randomly insert, delete, or cause frameshift mutations in the sequence being edited, which can dramatically alter gene function.
Cas-CLOVER has been validated to have little to no off-targets due to its dimeric nature. The Cas-CLOVER gene editing system shown in Figure 2 utilizes a catalytically inactive Cas9 protein fused to the Clo51 nucleus domain, which work as monomers recruited by a pair of guide RNAs (gRNA) to introduce targeted mutations. When both subunits are properly recruited to the target-site, dimerization and activation of the Clo51 nucleus domain occurs, leading to targeted gene disruptions.
Figure 2: Cas-CLOVER gene editing system
The Importance of Adaptable, Next-Generation Gene Editing Tools
The growing global population increases the threat of climate change and global pandemics, putting excess pressure on our food and medicine supply chains. Genetic modifications of plants and microbes could be the answer to more productive and resilient agricultural and healthcare supply systems. Traditional crop breeding techniques are blunt, time-consuming instruments compared to the speed and precision of today’s molecular tools.
Next generation CRISPR-Cas technologies like Cas-CLOVER allow for scarless gene editing, meaning no heterologous genes are left in the plant genome producing a non-GMO crop. Additionally, Cas-CLOVER has not only been validated in plants via deletion of RDR6 in tobacco, but also in yeast and CHO cells. The Cas-CLOVER system is therefore not only precise, but robust and adaptable, easily used across model systems to help researchers advance their work.
Interested in seeing if Demeetra’s molecular tools will work in your model system? Learn more about our research and how our team can help move your studies forward by contacting us today!