Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs.

Before the gene scissors CRISPR/Cas is able to recognize and change target areas of the genome of plants, it must first be introduced into their cells. Scientists have various methods of old genetic engineering to choose from: a ring-shaped piece of DNA (a so called plasmid) containing foreign DNA can be introduced into the cells by the root bacterium Agrobacterium tumefaciens to form the gene scissors. Another method that is often used is particle bombardment with a gene gun, through which small DNA-coated metal particles are shot into the plant cells. As an intermediate step, a genetically modified plant is often produced with the DNA encoding the gene scissors being integrated in the genome. Scientists want to avoid this intermediate step, as unwanted changes in the genome can be generated during that step.
In the study, a new method is presented that introduces the CRISPR/Cas system into plant cells by using RNA viruses. One advantage is that the transmitted RNA is not unintentionally integrated into the genome of the plants by the RNA viruses, as DNA could be. However, there is one major drawback: the DNA encoding the gene scissors is too big and the RNA viruses do not have the capacity to transfer such a large DNA fragment. There are only a few cases in which the DNA encoding the gene scissors has been successfully introduced into plants with the help of RNA viruses. The efficiency of the gene scissors in generating a change in the desired target area is very low, and inheritable changes are only rarely made.
The scientists are trying to solve this issue: They decouple the transmission of the actual gene scissors (CRISPR/Cas) from the transmission of the guide RNAs (gRNAs), which enable the gene scissors to identify the target regions on the genome. Since the instructions for the gRNA are much shorter than the instructions for the nuclease, this element can be introduced into the cells by the RNA virus.
They use transgenic tobacco plants that already have the DNA encoding the CRISPR/Cas9 gene scissors inserted into their genome. The gRNAs are introduced into the plants via the tobacco rattle virus and recognize a gene called phytoene desaturase (PDS). Silencing of this gene causes the leaves of plants to become lighter.
In addition, further RNA elements were introduced that are intended to intensify the effects: To increase efficiency, the gRNAs were fused to so-called mobile RNA sequences, which bring the gRNAs from the site of infection to the growth area of the shoot generating more heritable changes.
The result: it was shown that new leaves with bleached areas were indeed growing, showing that the PDS gene had been silenced. The frequency with which mutations occur in the newly growing leaves is over 80%. Up to 65% of the seedlings derived from the infected plants carry a mutation in the target gene.
The use of RNA viruses to introduce CRISPR/Cas is still at the beginning, as scientists have to work with transgenic plants that carry the DNA encoding the gene scissors. The goal is to introduce both the gene scissors and the gRNAs with the help of the RNA viruses avoiding the intermediate step via a transgenic plant. The method described here does not offer an alternative to the most common transformation methods using Agrobacterium or the gene gun.