In plants, DNA methylation is one of the major epigenetic marks regulating the transcriptional activity of many genes. A specific locus can be efficiently silenced by small RNAs that induce de novo DNA methylation by RNA-directed DNA methylation pathway (Přibylová et al. 2019). Together, DNA methylation and transcription were shown to influence the mutational outcome of CRISPR/Cas9 in plant cells (Přibylová at al. 2022). Although many aspects of the interactions between DNA methylation, transcription, Cas9 cleavage and subsequent DNA repair have been described, the whole interaction network is far from being fully understood.

The proposed doctoral project focuses on the interrelationships between the above processes. The work will be done on tobacco BY-2 cell lines, which proved to be a suitable model for studying de novo DNA methylation and editing by CRISPR/Cas9. We offer an up-to-date topic that includes modern experimental approaches, such as the generation of genetically modified cell lines, chromatin-immunoprecipitation, and advanced bioinformatic analyses. We expect applications from highly motivated students capable of independent work. Only applications with a motivation letter directly addressing the project subject and demonstrating a genuine interest and understanding of the topic will be considered for selection.

Five relevant publications of the research group:

Přibylová A, Fischer L, Pyott DE, Bassett A, Molnar A (2022) DNA methylation can alter CRISPR/Cas9 editing frequency and DNA repair outcome in a target-specific manner. New Phytologist 235: 2285-2299, doi: 10.1111/nph.18212.

Čermák V, Tyč D, Přibylová A, Fischer L (2020) Unexpected variations in posttranscriptional silencing induced by differentially produced dsRNAs in tobacco cells. BBA – Gene Regulatory Mechanisms 1863(11):194647, doi: 10.1016/j.bbagrm.2020.194647.

Přibylová A, Čermák V, Tyč D, Fischer L (2019) Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells. Epigenetics & Chromatin 12: 54, doi: 10.1186/s13072-019-0299-0.

Čermák V; Fischer, L (2018) Pervasive read-through transcription of T-DNAs is frequent in tobacco BY-2 cells and can effectively induce silencing. BMC Plant biology 18: 252, doi: 10.1186/s12870-018-1482-3.

Nocarová E, Fischer L (2009) Cloning of transgenic tobacco BY-2 cells; an efficient method to analyze and reduce high natural heterogeneity of transgene expression. BMC Plant Biology 9:44, doi: 10.1186/1471-2229-9-44.

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