Targeted engineering of plant centromeres
General Genetics and Functional Genome Biology
Final Report Abstract
The formation of de novo centromeres by LacI-based targeting of CENH3 to LacO sites in generative tissues, especially during meiosis was tested. The stable activity of de novo centromeres would be required for the transmission of a minichromosome to the next generation. Unfortunately, we were not able to confirm the presence of de novo centromers that met the full set of criteria, that is, co-localization of CENH3 with LacO repeats and formation of anaphase bridges, in generative tissues, especially during meiosis. We therefore concluded that this system is either insufficient to form de novo centromeres or the amount of CENH3 enrichment at LacO sites is below the level of detection. To achieve our goal by an alternative strategy based on the newly developed CRISPR-dCas9 system, we fused the sequence encoding Arabidopsis CENH3 to the C-terminal end of dCas9 codon optimized for dicotyledonous plants. dCas9-CENH3 was targeted to a repetitive DNA sequence with the 45S-rDNA loci of Arabidopsis by designed a complementary sgRNA. We found CENH3 localization at target loci detected by immunofluorescence labelling of CENH3 coupled with FISH against 45s rDNA loci in interphase nuclei from vegetative tissue. But, we were not able to observe a targeted localization of CENH3 at 45s rDNA during meiosis. Similarly, this would be required for transmission of a minichromosome to the next generation. To confirm whether the dCas9-CENH3 protein was present in generative tissues, we performed a Western-blot aiming to detect the dCas9-CENH3 protein in flower buds. A very low amount of the actual dCas9-CENH3 protein was detected in flower buds. We therefore concluded that this system is able to target CENH3 to a specific locus throughout interphase in vegetative tissue but yet insufficient to generate an active centromere during meiosis. Simultaneous we trialed ‘CRISPR-FISH’ by selecting the A. thaliana centromere-specific 180bp repeat as a target sequence and developed a Cas9 fluorescent protein fusion construct aiming to label centromeres in living Arabidopsis cells. This preliminary experiment using SadCas9- TurboFP635 enabled the detection of distinct foci in nuclei of living root cells of Arabidopsis that resembled the distribution of centromeres. To test the functionality of CRISPR-dCas9 for live cell imaging in plants, we imaged the telomeres of Nicotiana benthamiana in leaf cells.
Publications
- (2017) Live-cell CRISPR imaging in plants reveals dynamic telomere movements. The Plant journal : for cell and molecular biology 91 (4) 565–573
Dreissig, Steven; Schiml, Simon; Schindele, Patrick; Weiss, Oda; Rutten, Twan; Schubert, Veit; Gladilin, Evgeny; Mette, Michael F.; Puchta, Holger; Houben, Andreas
(See online at https://doi.org/10.1111/tpj.13601) - (2015) Engineering of plant chromosomes. Chromosome Res, 23, 69-76
Michael Florian Mette & Andreas Houben, A.
(See online at https://doi.org/10.1007/s10577-014-9449-1) - Engineering of Arabidopsis thaliana chromosomes, 2015, Plant Science Student Conference, Germany
Steven Dreissig, Michael Florian Mette, Andreas Houben
- Approaches to CRISPR/Casbased chromosome engineering in plants, 2016, Tri-National Arabidopsis Meeting, Austria
Steven Dreissig, Andreas Houben, Michael Florian Mette
- Investigating the effect of CRISPR-dCas9-based binding of CENH3 to non-centromeric loci, 2016, International Chromosome Conference, Brazil
Steven Dreissig, Michael Florian Mette, Andreas Houben
