Gene editing has progressed in leaps and bounds in the past few years, particularly through the expansive use of CRISPR and the dozens of variant tools made from it that have spawned. But, at the same time, many of those tools focus on fixing individual problems or limitations in the original CRISPR systems and they often themselves fall into the same traps of usage. In short, editing a genome is something that often can only be done on a single gene or a string of connected genes, with disparate genes across a genome being difficult to address in a single action. There are many cases where multiple generations of edited plants are needed to get the final outcome trait that was desired from the very beginning. 

Some exceptions have occurred, with tools being able to affect large scale expression of genes and epigenetic controlling of on or off gene mechanics. Though these commonly suffer from other problems, including efficiency losses within a single plant or a lack of upkept activity over time within an overexpressed plant gene. Many scientists and research teams have been trying to get around these limitations and create a multi-functional tool that can edit freely and manipulate expression on a higher level than just single gene activation. And let it not be said that they haven’t been successful. 

Introducing The Combo

Today’s discussed new advancement is far from the only one to take such a step, but it’s another with proven success and great potential for future usage on plants. With the end goal being as usual to allow for CRISPR, at least for its use in agriculture, to produce crops that have new traits and resistances to help stabilize yields and provide protections for the future climate and other related struggles we will be facing. 

With that all said, let’s discuss CRISPR-Combo. Created by a research team at the University of Maryland, College Park, their focus was on making a high efficiency tool that could simultaneously cause gene edits and transcriptional up and down regulation of genes to occur, all within the same package. To accomplish this, they engineered a complex scaffold of single guide RNAs (sgRNAs) that would be able to control how the CRISPR protein complex functioned, without relying on the fused Cas9 proteins regulating that functionality. This allows the Cas9 and sgRNAs to act independently of each other and forms the basis for the Combo system so that editing and regulation can occur in tandem. 

Their past studies on the tool had shown that it was able to work in this manner on rice and tomato cells. They next wanted to do a myriad of tests on the model organism Arabidopsis and on energy and crop plants such as poplar and rice. The goal was to make desired trait edits to the plants while also overexpressing genes related to flowering and regeneration so that the growth speed would be enhanced, with that lengthy growth period being one of the primary difficulties with large scale gene editing studies. 

The Three Experiments

Their first target was that very accelerated breeding issue and their tests in Arabidopsis included upregulating a florigen gene that caused early sprouting and flowering to occur. This had side benefits of not only being a visible phenotypic response to show differing levels of expression from Combo, but also made it incredibly easy to pick out which seedlings had been successfully transformed in the batch. And screening for transformants is the bane of many a gene editing process. They found that they had a 92% accuracy rate in screening afterward and the groups that they were testing with simultaneous editing of herbicide resistance genes showed a greater than 50% success rate on that front as well. Investigation of the genome changes also found no evidence of off-target effects in either case. With that proven in Arabidopsis, they then moved on to poplars and their enhanced regeneration testing. 

It  is common for plants in the regeneration research field to be resistant to those processes, which also causes the speed of gene editing testing to slow dramatically. The researchers in this case wanted to alter expression of the so-called morphogenic genes in order to enhance their regeneration speed from cells to full plants. They hoped that overexpression of particular genes, including those in the well known WUSCHEL family responsible for shoot and flower development, would speed up how quickly the stems and roots of the poplar plants would form. Amazingly, 100% of the lines tested with CRISPR-Combo saw gene editing occur, with over 75% having it happen on both copies of the genome to make homozygous poplars for the edited gene. They also found that accomplishing a 200-fold expression increase of the genes reflected a massive increase in growth speed.

The third and final experiment the team conducted was on improving rice plants by activating morphogenic genes. Their initial tests on calluses to improve regeneration rates into full plants was majorly successful, producing even more regenerants than they expected when starting the experiment. This led the scientists into trying to use morphogenic genes to also enhance overall efficiencies for gene editing using CRISPR-Combo. With the larger goal being able to do so without requiring external application of hormones to better trigger regeneration as so many other methods are forced to use. 

They saw results in their testing using a hormone free culture medium, having over 35% of the calluses regenerate into full plants and each callus alone seeing two to five plants emerge during the process. Their control group without using the gene activation saw only two out of 100 calluses regenerate appropriately. An additional interesting result was that the actual expression of the target morphogenic gene in the regenerated plants did not see increased RNA production but still kept the same improved growth traits. This meant that the expression alterations from Combo had continued to the downstream gene regulators for growth and development and were heritable by the following generation.

A New Tool And The Future

The researchers ended their study by noting the massive impacts on speed breeding that CRISPR-Combo enabled by reducing the time it takes to reach flowering, as shown in the Arabidopsis model. Also that their tool had high efficiencies and the selection of transformed mutants could be done between wild type and transformants just from visible inspection.  Lastly, while not noted previously, the use of the floral dip method and Agrobacterium transformation as the medium for using CRISPR-Combo meant that a dedicated growth chamber was unneeded when producing the plants. 

Beyond speed breeding, their advances in plant regeneration from cell and tissue cultures have massively reduced the need of additional inputs and hormonal controls to ensure proper activation of the CRISPR tool. This in turn revealed the connections between gene editing and gene expression in the plant genome and how their combination through Combo further enhances the selection capabilities for gene edited plants thanks to expression having visual changes to the growth of the plants. This was shown through the Arabidopsis having the very apparent early flowering trait. 

As can be seen from the multiple experiments the research team conducted in this study, the possibilities for CRISPR-Combo and its joint editing and expression methods can be used in any variety of ways with any number of other plants and maybe even beyond that. A dual purpose genetic modification tool such as this has incredible potential that can’t be overstated. We’ll just have to watch and see where the scientific community takes it from here. 

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Photo CCs: Poplar Trees falling leaves-3 (17301340386).jpg from Wikimedia Commons

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