Genome editing: il futuro (prossimo) del miglioramento genetico delle piante

Andrea Cavallini [Dipartimento di Scienze Agrarie, Alimentari e Agro-ambientali dell'Università di Pisa]
Tommaso Giordani [Dipartimento di Scienze Agrarie, Alimentari e Agro-ambientali dell'Università di Pisa]
Lucia Natali [Dipartimento di Scienze Agrarie, Alimentari e Agro-ambientali dell'Università di Pisa]

Genome editing, or genome editing with engineered nucleases, is a technology that, using engineered nucleases, allows site-specific single-base mutations or the insertion, deletion or replacement of DNA sequences in a specific site in the genome of an organism. Genome editing is based on the induction of double strand breaks (DSBs) in the DNA in the locus of interest to introduce mutations in that locus. In fact, after DSB induction, the damage will be repaired by processes (the non-homologous end joining and/or the homology-directed repair), that occur naturally in the cells and during which mutations may occur.

DSBs can be induced by different nucleases, all capable of specifically recognising a locus in the genome. The most promising is the CRISPR/Cas system, for ease of designing nucleases with sequence specificity and for the fact that it can be used in nearly every organism. In the CRISPR/Cas9 system, the recognition of the DNA sequence to be modified is operated by an RNA sequence. After successful DNA DSB, the cell proceeds with the repair of DNA. Generally, the cell uses non-homologous end joining, which produces substitutions, insertions and deletions of nucleotides in the damaged DNA site, and usually leads to loss of function of the target gene. When using this mode, the genome editing can be considered a biological site-specific mutagenesis, different from the mutagenesis induced by physical or chemical agents which randomly induce mutations through the entire genome. On the contrary, when homology-directed repair is involved, genome editing can be considered a predetermined biological mutagenesis that modifies or corrects the target gene in the sense determined by the investigator.

Applying genome editing to plants requires also ancillary technologies, according to the species and cell types. First, in vitro culture techniques, especially protoplast cultures, might be necessary for the production of cells that can be subjected to the nuclease treatment. Then, transformation vectors (Agrobacterium, viruses or biolistic methods) are needed to enable the transfer of the components required for genome editing to the plant cell. The vectors may be stable or transient; in the latter case, both the possible cytotoxicity of constitutively expressed nucleases and the production of transgenic plants would be avoided.

Concerning the first results obtained using this technology, mutations in target genes of cultivated plants were obtained mostly through non-homologous end joining for traits related to morphology, quality and to the resistance to pathogens and herbicides, in both herbaceous and woody species. Results were also reported exploiting the homology-directed repair. Overall, the genome editing technology proved suitable to introduce precise and predictable gene mutations directly into elite cultivars, reducing the duration of traditional crossing and backcrossing breeding, with the possibility to modify more than one genes per experiment.

Although many advances in genome editing technology have been achieved in recent years, some technical problems remain to be solved, including the need for increasing the efficiency of the system, the production of off-target mutations, the influence of chromatin structure on the editing efficiency, the possible side effects on genes lying close to target genes and the efficiency of the technology in polyploid species (where many copies of target genes occur).

In conclusion, the CRISPR/Cas system has emerged as the most important tool for the future of genetics because of its simplicity, versatility and efficiency. It will have a major impact on both basic and applied research and will be used to produce cultivars with improved disease resistance, with a higher nutritional value, and able to survive climate changes, more suitable as bioenergy crops, producing useful chemicals and biomolecules.

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