Jumping genes for efficiency
At the moment there is no efficient way to precisely insert large chunks of DNA in a plants genome. Now American researchers show a way in Nature that is ten times as efficient than current methods.
Sometimes you like to insert a gene in order to find out what it does. Sometimes you do it because you like to make a crop disease resistant. But whatever the reason, there is no efficient way that inserts a gene in the preferred location in the genome.
When you think about precise edits of the genome you probably think about CRISPR-Cas. This system can with great precision make a cut in the DNA. Useful for single base edits. But integrating larger chunks of DNA is a different story. To change that the researchers look in the direction of jumping genes.
Combining CRISPR-Cas9 with the Pong transponases increased the efficiency 10 times
With help of transponases jumping genes cut themselves out of the genome and intergrate themselves in a new spot in the genome. Where exactly depends on the preferences of the jumping gene. But it can be at a location where the DNA is broken. Of note is that during this jumping the transponases protect the DNA against degradation.
The researchers decided to study if was possible to increase the efficiency of integrating large chunks of DNA in the genome by combining CRISPR-Cas and jumping genes. They decided to use in the rice jumping gene mPing and its Pong transponases ORF1 and ORF2.
In order to test if this combination could work the researchers first created an Arabidopsis plant with mPing. When they added CRISPR-Cas9, ORF1, and ORF2, then the researchers noticed mPing jumping to the by the CRISPR-Cas guide-RNA designated spot. This occurred with great precision, which increased when the enzymes Cas9 and ORF2 were linked. But this linking came at a cost. Was the efficiency without linking around the 35.5%, was this with linking around the 6.7%. Still higher than the current precision methods which have an efficiency between the 0.24 and 4.8%.
The researchers expect the system to work in all plants, except in rice
Subsequently the researchers tested if the system was still working with a foreign gene located within mPing. This turned out to be. Although, an extra 8994 basses also reduced the efficiency a little bit.
The big question was does the system also work on other plants than the up till now used Arabidopsis. To find this out the researchers turned to soyabeans. In which the system also appeared to work. Although some adjustments were needed. Like a longer linker between Cas9 and ORF2.
The researchers expect that the new system, that they named TATSI, will work in other plants as well. With one exception, rice, because each species suppresses its own jumping genes. Therefore fore rice an jumping gene from another species will be required.
Literature
Liu, P., Panda, K., Edwards, S.A. et al. Transposase-assisted target-site integration for efficient plant genome engineering. Nature 631, 593–600 (2024). https://doi.org/10.1038/s41586-024-07613-8
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