Pacher, Michael

Puchta, Holger

Schmidt, Carla

English

JKI Open Journal Systems (Julius Kühn-Institut)

11th Young Scientists Meeting 2018, Braunschweig, Germany, November 14-16 Schmidt et al. Efficient induction of inversions in plant genomes using the CRISPR/Cas system Carla Schmidt, Michael Pacher and Holger Puchta Karlsruhe Institute of Technology, Botanical Institute, Karlsruhe E-mail of corresponding author: carla.schmidt@kit.eduCRISPR/Cas mediated genome engineer-ing in plants has mainly concentrated on the knockout of genes and the induction of deletions. Sequence inversions occur often during plant genome evolution - also in related cultivars - making the in-verted regions inaccessible for plant breeders as a result of fixed linkages. Thus, it is important to set up technolo-gies that allow us to induce inversions within chromosomes in a directed and ef-ficient way in plants. In the current research, we induced two DSBs in a distance of about 3 - 18 kb using the Cas9 protein from Staphylococcus au-reus (SauCas9) for efficient DSB induction on four different loci in the model plant Arabidopsis thaliana. The expression of the Cas9 nuclease was controlled by the constitutive PcUbiquitin4-2 promoter from Petroselinum crispum (PcUbi4-2) and by the egg-cell specific promotor (EC-P) of A. thaliana. The approach, driven by the PcUbi4-2 promotor, was analysed in T1 using digital droplet PCR (ddPCR) to quantify the amount of deletions and in-versions in wildtype and ku70-1 mutants.It was possible to generate deletions with frequencies up to 7 % and inversions up to 2 %. Additionally, we defined via deep sequencing the patterns of junction for-mation in wildtype and ku70-1 mutants. Like for deletions, in the majority of cases for inversions re-joining of the cut junc-tions occurs without further mutations. Surprisingly, in plants deficient in KU70, which is essential for classical non-ho-mologous end joining (NHEJ), inversion induction is enhanced. However, most junctions - that often contain micro ho-mologies - are imperfect with insertions and mainly deletions. Using the egg cell specific expression we were able to in-duce heritable inversion formation at dif-ferent loci and at distances between 3 and 18 kb in the percent range. By screening individual lines heritable inver-sion events up to the 10 % range can be regularly achieved. Most of the events contained the inversion with scarless junctions and without any sequence change within the inverted region making the technology attractive for applications in crop plants. 46 Berichte aus dem Julius Kühn-Institut 200

Efficient induction of inversions in plant genomes using the CRISPR/Cas system

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Efficient induction of inversions in plant genomes using the CRISPR/Cas system

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