Belgium: CRISPR-based tissue-specific knockout system to help generation of specific plant mutations

A team from the VIB-UGent Center for Plant Systems Biology, led by Drs. Thomas Jacobs, Moritz Nowack, and Tom Beeckman has devised a CRISPR-based tissue-specific knockout system, CRISPR-TSKO. This system enables the generation of specific mutations in particular plant cell types, tissues, and organs. The efficiency of CRISPR-TSKO opens new avenues to discover and analyze gene functions during the life of plants while avoiding the effects of system-wide loss of gene function. The work will be published in the authoritative journal Plant Cell.

One, two, three, knockout
Knocking out genes is a great way to learn what they do. After all, if you prevent a gene from doing its job and you notice changes, it’s very likely the gene has something to do with it. There is a caveat, though. If a scientist mutates a gene that is required for growth and/or reproduction, the mutant plants are often very sick or even die. This really prevents the investigation of so-called essential genes using the knockout approach. Additionally, some genes have different functions in different parts of a plant, so the effect of a knocking out a gene in one part could mask its function elsewhere. One solution to this limitation is the generation mutations in specific plant tissues.

Getting specific with CRISPR
Now, researchers led by Drs. Thomas Jacobs, Moritz Nowack, and Tom Beeckman (VIB-UGent Center for Plant Systems Biology), have developed a tissue-specific knockout system in plants based on CRISPR, CRISPR-TSKO.

After delivering the system into the plant Arabidopsis, they could quickly assess whether the plant cells had the desired mutations. For this, they had help from the VIB Flow Core where thousands of cells from seedlings were sorted. After the sorting, they confirmed that, indeed, the cells they had targeted carried the mutations they had hoped to elicit.

Dr. Jacobs remembers the moment the work’s relevance dawned on him: “After the first proof-of-concept experiment. A number of individual lines were completely knocked out for GFP expression only in the lateral root cap. It was immediately clear that this was going to work.”

With this new technology, the functions of specific genes can be investigated in specific plant tissues at specific times in a plant’s development. One of the future aims of the team is to investigate genes that are essential for plant life, a feat that is currently very difficult to achieve. 

Dr. Jacobs muses about the future of this research: “We would really like to develop a larger collection of tissue-specific promoters and test these with numerous genes that are essential for plant growth and development that cannot be investigated otherwise. It is really challenging to work with essential genes, and I think this system can be used to challenge some long-held theories on the function of certain ones.”

Source: VIB


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