09.04.2020

Better Cas9 efficiency for gene knock-out in plants.

A team led by Sylvestre Marillonnet recently published a preprint of a study in which they tried to optimize the Cas9 enzyme for gene editing in plants. In plants, site-targeted mutations are usually obtained by stably transforming a Cas9 expression construct into the plant genome. The efficiency with which mutations are obtained in genes of interest can vary considerably. In their optimization effort, the researchers found that the Cas9 gene codon usage had some effect on Cas9 activity and that two nuclear localization signals work better than one. However, the most important impact on the efficiency was obtained by addition of 13 introns into the Cas9 coding sequence, which dramatically improved editing efficiencies. None of the primary transformants obtained with a Cas9 lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of transformants generated with intronized Cas9 displayed mutant phenotypes. The intronized Cas9 was not only found to be effective in Arabidopsis but also in other plants such as Nicotiana benthamiana.

Note: A preprint is a version of a paper that precedes formal peer review and publication in a peer-reviewed journal and is published on dedicated preprint platforms.

Originalpublikation:
Addition of Multiple Introns to a Cas9 Gene Results in Dramatic Improvement in Efficiency for Generation of Gene Knockouts in Plants. Ramona Grützner, Patrick Martin, Claudia Horn, Samuel Mortensen, Erin J. Cram, Carolyn W. T. Lee-Parsons, Johannes Stuttmann, Sylvestre Marillonnet bioRxiv 2020.04.03.023036; doi: https://doi.org/10.1101/2020.04.03.023036