If one is trying to find new and unique traits and abilities in any organism, then the regular span of generations takes far too long in the natural world. Instead of relying on evolution by natural selection, directed evolution by human hands works much better and can be accelerated in multiple ways. So far, the vast majority of such methods have only been developed to work in microorganisms, which can be more readily induced to evolve changes in fast generations. But if someone wants to create a more complicated biomolecule en masse, then a more complex host must be used that can follow such a biochemical pathway. 

There are options available for conducting such evolution in eukaryotic cells, such as setting up screens after mutagenesis and picking out specific mutant cells after exposure to or specific directed alterations in genes of interest. But, until now, the gene editing powers of CRISPR have only minimally been used to evolve the genomes of complex organisms, especially when it comes to plant genomes. We’ve previously discussed CRISPR-based tools such as EvolvR or epimutagenesis or even something rather relevant to today’s paper in the RNA splicing control tool of CasRx. But they’ve all been for broad-based options and not specifically for plant usage. 

CRISPR Rice Evolution

A research team at KAUST has now taken up the task of creating a platform for using CRISPR in this sort of manner in plants. The plan was to make a platform that can cause double-strand breaks in DNA for a specific gene at all the possible coding portions of that gene. They chose to test this creation by focusing on a particular protein involved in providing resistance to inhibitors of splicing and processing RNA molecules known as SF3B1. The usual mechanism of Agrobacterium infection was done in order to transfer the CRISPR complex into the rice plant being used as a model.

Once incubated with the bacteria, the plants were put under selective pressures to rapidly cause evolution due to the double-strand breaks the CRISPR complex was actively making in their genomes. Those that survived the pressures were then sequenced to determine what exactly the mutations had caused in the inhibitor resistance gene. The hope was that variants of the SF3B1 inhibition resistance would manifest and provide resistance to an even broader range of such inhibitors. 

Some mutant seedlings were created that were still RNA splicing functional, but seemed to have changes that affected the ability of drugs to bind to them. Across all the mutants seen, there were none living that had a knockout version of SF3B1, implying that loss of function of this gene is lethal beginning in the embryonic stage and is needed in order for seedlings to be viable.

Diversifying Mutant Resistance

The research team next went broader and tested if changing the protein domains for SF3B1 that interacted with drugs would allow for the creation of the desired variants. They specifically focused on finding variants resistant to the inhibitor herboxidiene. Their CRISPR platform was then able to successfully produce those mutants and they were confirmed to be inheritable mutations passed on to future generations. Though there is no physical phenotype to notice that there has been a change to RNA splicing genes, so only sequencing can show the mutant from the wild-type. 

Through several experiments, the team was able to confirm their formation of a mutant strain they called SF3B1 SGR4 that was resistant to herboxidiene binding to it, while the wild-type doesn’t have resistance to this splicing inhibitor. Thus, they were able to confirm that their platform works and that CRISPR can be employed for genome evolution in plants to improve and diversify the traits used across agriculture. 

Broadening The Trait Horizon

The scientists hope that the previously mentioned other systems are also set up in the future to work in such a platform for organisms outside of plants and that the mutation space can be well populated by combining CRISPR with selective pressures. While this is only a proof of concept that their design does indeed work, there is now the need to apply it to plants across the board and truly develop those trait variants that can benefit different areas of plant research.

Press Article

Study Article

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