Modification of ethylene sensitivity in ornamental plants using CRISPR/Cas9
Ethylene sensitivity has for long been of interest in improving ornamental plants e.g., Kalanchöe and Campanula. We aim to investigate changes in ethylene sensitivity in economically important ornamental plants by targeting genes in the ethylene pathway using the novel precise genome editing tool; the Clustered Regularly Interspaced Palindromic Repeats (CRISPR) RNA guided Cas9 DNA nuclease (CRISPR/Cas9). CRISPR/Cas9 may be employed to introduce targeted double-stranded breaks (DSBs) at desired sites in the host genome. The DSBs will be repaired by the non-homologous end-joining (NHEJ) repair mechanism which often results in small indels and consequently gene knockout. The CRISPR/Cas9 system consists of a protein DNA nuclease (Cas9) which is guided to the target sequence by a small RNA molecule (sgRNA) that recognizes a 20 bp target sequence in the genome situated immediately downstream of a 3 bp protospacer adjacent motif (PAM). The sgRNA confers the sequence specificity of the CRISPR/Cas9 complex and may thus be designed to target virtually any sequence, a feature that has made it the method of choice within precise genetic engineering. Although most research with CRISPR/Cas9 has been conducted in prokaryote and mammalian cells, steps have been taken to implement the system in plants. Proof of function has been obtained in various plant species e.g., Arabidopsis, wheat, soybean and orange which makes it plausible that this technique could be applied to ornamental plants as well. The CRISPR/Cas9 system will be delivered using Agrobacterium tumefaciens and explant regeneration of tissue cultures to create stable transformation and mutation events.
Kemp, O., Favero, B.T., Hegelund, J.N., Møller, S.R., Müller, R., Petersen, B.L. and Lütken, H. 2017. Modification of ethylene sensitivity in ornamental plants using CRISPR/Cas9. Acta Hort. (ISHS) 1167:271-280
senescence, genome editing, Kalanchöe, Campanula