Crop Biotech Update

Enhanced CRISPR-Cas9 Enables Stable Insertion of Large Genes

May 15, 2024

Local genetic modification using the CRISPR-Cas gene editing method in the leaves of the Australian tobacco N. benthamiana. Green fluorescent spots represent leaf cells where a large gene segment was successfully inserted. This experiment used these Cas9 variants: inactive (left), active (center), and Cas9 with an exonuclease (right). Only the use of the exonuclease causes a strong increase in correct gene insertion. Photo Source: Tom Schreiber, IPB

Scientists at the Leibniz Institute of Plant Biochemistry (IPB) have successfully inserted large gene segments into the DNA of higher plants very efficiently in a stable and precise manner. The research team optimized CRISPR-Cas9, and the improved method offers great opportunities for the targeted modification of genes in higher plants, both for breeding and research.

CRISPR-Cas has enormous potential for the targeted modification of individual genes. However, this does not apply to all kinds of genetic modifications. While CRISPR is ideal for knocking out genes, they do not work well for precisely inserting genes or replacing gene segments. Targeted insertion of genes into the DNA of higher plants has only been successful in rare individual cases.

To increase the success rate of gene insertion, the Halle scientists equipped CRISPR-Cas with an additional enzyme, an exonuclease to alter the DNA cleavage sites created by the genetic scissors. The cell's internal repair enzymes can no longer recognize and mend the DNA damage. The DNA segment to be inserted by CRISPR-Cas would therefore have enough time to integrate itself at the correct position through another, very precise, cellular repair mechanism. The Halle scientists tested various exonucleases of viral, bacterial, plant, and human origin for their ability to increase the number of precise gene insertion events. They introduced the genetic scissors with the corresponding exonucleases and a gene X segment into the leaf cells of the tobacco plant Nicotiana benthamiana which had been previously equipped with a gene for a green fluorescent marker.

The green marker can only be produced when the missing gene section of X is precisely reinserted using CRISPR/Cas, thus repairing gene X. Two of the exonucleases tested proved to be particularly effective. Using these, the team from Halle achieved 38 times more perfect gene insertion events than with CRISPR-Cas alone.

For more details, read the news release on the IPB website.

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