Variability of DSB repair in plant development and genome evolution, at molecular and chromosomal level

• Applicant: Professor Dr. Ingo Schubert
• Subject Area: Plant Genetics and Genomics ; Plant Cell and Developmental Biology
• Term: from 2012 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 214652223

DNA double-strand breaks (DSBs) represent the most complex and hazardous DNA lesion. Consequently, several repair pathways with different requests on homologous template sequences, and different precision of outcome, have evolved.During the ongoing project, we found a strategy to trace all DSB repair pathways in barley by combination of cytogenetics and amplicon sequencing from transgenic target sequences. Surprisingly, the majority of DSBs were repaired during S/G2 phase of cell cycle via reciprocal exchange using the sister chromatid as a template. In addition, efficient DSB-induced gene targeting was for the first time achieved for barley. Based on these results, we established an approach to monitor at the target site simultaneously the variants of NHEJ which use various lengths of microhomology, as well as HR, resulting either in restitution or in conversion, deletion, insertion, or combined mutation types. Applying a novel sequencing technology (Pacbio), we will now compare the same reporter genes in A. thaliana and in barley to find out potential differences in repair capacity during different developmental stages in both species.At the same time we will test for possible differences between large and small genomes that might drive genome size evolution, via a bias towards more and/or longer deletions in small genomes. Furthermore, we intend to compare the data obtained for transgenic reporter genes with DSBs generated by CRISPR-Cas9 RNA-guided nucleases on Arabidopsis endogenous sequences mimicking the sequence arrangement of reporter genes (for preferential detection of either SSA or SDSA). The effect of CRISPR-Cas9 nuclease-induced DSBs and CRISPR-Cas nickase-induced SSBs on the spectrum of repair pathways will be tested at the molecular (sequencing) and the chromosomal level (SCE, chromosome aberrations) at the same reporter genes and on repetitive endogenous sequences in barley (SCEs). Additionally, the effect of DSBs at homozygous versus hemizygous loci on the origination of asymmetric chromosomal translocations will be investigated as well as the proportion at which these rearrangements occur before or after replication.

• DFG Programme: Research Grants