Unser 10. Leibniz Plant Biochemistry Symposium am 7. und 8. Mai war ein großer Erfolg. Thematisch ging es in diesem Jahr um neue Methoden und Forschungsansätze der Naturstoffchemie. Die exzellenten Vorträge über Wirkstoffe…
Omanische Heilpflanze im Fokus der Phytochemie IPB-Wissenschaftler und Partner aus Dhofar haben jüngst die omanische Heilpflanze Terminalia dhofarica unter die phytochemische Lupe genommen. Die Pflanze ist reich an…
Geschmack ist vorhersagbar: Mit FlavorMiner. FlavorMiner heißt das Tool, das IPB-Chemiker und Partner aus Kolumbien jüngst entwickelt haben. Das Programm kann, basierend auf maschinellem Lernen (KI), anhand der…
Schreiber, T.; Tripathee, S.; Iwen, T.; Prange, A.; Vahabi, K.; Grützner, R.; Horn, C.; Marillonnet, S.; Tissier, A.;DNA double strand breaks lead to de novo transcription and translation of damage-induced long RNAs in plantabioRxiv(2022)DOI: 10.1101/2022.05.11.491484
DNA double strand breaks (DSBs) are lethal threats that need to be repaired. Although many of the proteins involved in the early steps of DSB repair have been characterized, recent reports indicate that damage induced long and small RNAs also play an important role in DSB repair. Here, using a Nicotiana benthamiana transgenic line originally designed as a reporter for targeted knock-ins, we show that DSBs generated by Cas9 induce the transcription of long stable RNAs (damage-induced long RNAs - dilRNAs) that are translated into proteins. Using an array of single guide RNAs we show that the initiation of transcription takes place in the vicinity of the DSB. Single strand DNA nicks are not able to induce transcription, showing that cis DNA damage-induced transcription is specific for DSBs. Our results support a model in which a default and early event in the processing of DSBs is transcription into RNA which, depending on the genomic and genic context, can undergo distinct fates, including translation into protein, degradation or production of small RNAs. Our results have general implications for understanding the role of transcription in the repair of DSBs and, reciprocally, reveal DSBs as yet another way to regulate gene expression.
Preprints
Grützner, R.; Martin, P.; Horn, C.; Mortensen, S.; Cram, E. J.; Lee-Parsons, C. W. T.; Stuttmann, J.; Marillonnet, S.;Addition of Multiple Introns to a Cas9 Gene Results in Dramatic Improvement in Efficiency for Generation of Gene Knockouts in PlantsbioRxiv(2020)DOI: 10.1101/2020.04.03.023036
The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. 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 depending on specific features of the constructs, including the source and nature of the promoters and terminators used for expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency with which mutations could be obtained in target genes in Arabidopsis thaliana with the Cas9 nuclease, we have investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLS) and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on Cas9 activity and that two NLSs work better than one. However, the most important impact on the efficiency of the constructs was obtained by addition of 13 introns into the Cas9 coding sequence, which dramatically improved editing efficiencies of the constructs; none of the primary transformants obtained with a Cas9 lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of primary transformants generated with intronized Cas9 displayed mutant phenotypes. The intronized Cas9 was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.