Authors: V. Poobalan, R. Mohammed Aseef and K. Karthick
Research Scholar, Horticultural College & Research Institute, Tamil Nadu Agricultural University, Coimbatore, India
*Corresponding author: poobalanagri007@gmail.com
What is genome editing?
Genome editing , or genome editing with engineered nucleases ( GEEN) is a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of a living organism using engineered nucleases , or "molecular scissors." These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations ('edits').
Concept of genome editing
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The concept of genome editing was first demonstrated with the production of herbicide-tolerant crops. -
Treating weeds with herbicides, such as Phosphinothricin acetyltransferase (PAT), Acetolactate synthase (ALS), and Aryloxyalkanoate dioxygenase (AAD), has potential negative effects on crop production and yield. -
The prolonged application of these herbicide chemicals can lead to the development of herbicide-resistance in weeds. -
Anthocyanins are secondary metabolites that are present at high levels in many flowers, fruits, and vegetables, which act as major determinants of fruit ripening and quality. -
These compound are also utilized in the medicinal field due to their antioxidant properties (Hou et al, 2004).
Genome editing in vegetable crops
- Current genome editing approaches rely on the induction of cuts in double-strand DNA (DSB, double-strand breaks), which are then “repaired” through two different processes:
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Non-Homologous End Joining (NHEJ) or Homology-Directed Repair (HDR)
- Recently, the CRISPR/Cas9 system was successfully used to disrupt the gibberellin biosynthesis pathway gene GA4 to generate semi-dwarf plants in Brassica oleracea (Lawrenson et al., 2015).
- Similarly, in tomato, the gibberellin signaling gene PROCERA (PRO) was successfully modified by TALENs, resulting in a taller seedling phenotype (Lor et al., 2014).
- The phytohormone ethylene plays a vital role in plant growth and developmental processes such as fruit ripening and flower wilting. In tomato, the F-box genes Sl-EBF1 and Sl-EBF2 are actively involved in flowering and fruit ripening (Xie K Yang et al., 2013).
- Virus-induced gene silencing (VIGS) of either of the two Sl-EBF genes resulted in faster senescence and ripening in tomato
- Clasen et al. (2016) reported that TALEN-mediated silencing of the vacuolar invertase gene VInv prevented the accumulation of reducing sugars, brown pigments, and acrylamide compounds in cold-stored potatoes.
Applications of genome editing in crop improvement
The possibility to use genome editing tools either to achieve the crop ideotype by modifying major genes underlying key vegetative and reproductive traits, or induce the de novo domestication of wild relatives by manipulating monogenic yield-related traits, has been recently exemplified in tomato (Zsogon et al., 2017).
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TALE nucleases -have been used to knockout one DELLA gene (PRO) involved in negative regulation of gibberellic acid (GA) signaling. -
With the induction of frameshift mutations, loss of DELLA function and increased GA response, plants carrying two different mutant alleles (biallelic) or the same mutation in homozygous condition showed longer internodes than the wild type and lighter green leaves with smoother margins. -
In B. oleracea, CRISPR/Cas9 has been used to induce indel mutations in two regions of the BolC.GA4.a gene, which similarly to the homolog GA4 gene in Arabidopsis, is involved in gibberellin biosynthesis (Lawrenson et al., 2015). -
Some off-target mutations have been detected in another gene (BolC.GA4.b) which, compared with the GA4.a gene, showed two mismatches in target region 2. -
An alternative innovative approach for delivering editing reagents in plant cells has been recently reported in a number of species, including lettuce (Woo et al., 2015). -
In lettuce, the homolog of the Arabidopsis BRASSINOSTEROID INSENSITIVE 2 (BIN2) gene, encoding a negative regulator in the brassinosteroid (BR) signaling pathway, has been knocked out after transfecting PEG-treated protoplasts with a mixture of Cas9 and a sgRNA targeting the third exon of the gene. -
No off-target mutations were detected and plants regenerated via organogenesis from mutant calli transmitted the mutations to the progeny. -
Finally, the virus resistance of cucumber plants has been investigated after mutating the Eukaryotic translation initiation factor 4E (eIF4E) gene in two sites: in the first case, the gene was completely knocked-down, whereas, in the second, translation of two-thirds of the protein product was still possible (Chandrasekaran et al., 2016). -
Non-transgenic homozygous mutant plants showed either immunity or resistance to Cucumber vein yellowing virus (CVYV), Zucchini yellow mosaic virus (ZYMV), and Papaya ring spot mosaic virus- W (PRSV-W), although resistance breaking was observed in some cases.
Bottlenecks
The time and effort required for delivery of DNA to plant cells (i.e. getting necessary reagents across the cell wall) and the regeneration of plants containing the programmed changes. Production of plants is labour-intensive, slow and requires significant investment in technical expertise and training, which is why private sector companies such as Dow AgroSciences, DuPont Pioneer and Cellectis have been major contributors to research and development.
The necessary protection of intellectual property through the lengthy research and development (R&D) process required to bring new products to market means that there is some uncertainty about how genome editing has been taken up. Agricultural biotechnology giants appear to be awaiting developments in genome editing by the academic research base and translational research by smaller biotech firms.
To date, genome editing has been mainly focused on the control of single variants underlying qualitative traits. Quantitative variation is instead mediated by several nucleotides (QTN, quantitative trait nucleotides) with large and small effects on the phenotype. Editing quantitative traits is feasible once the availability of datasets of sequences and phenotypes will enable to discover large numbers of QTNs (Jenko et al., 2015).
A possible future achievement could be to perform a small number of edits on few QTNs with major effects. A further constraint in vegetable breeding is the manipulation of the reproductive system (e.g., apomixis and self-incompatibility). Such traits are under the control of several candidate genes (Hand and Koltunow, 2014; Yamamoto and Nishio, 2014) and genome editing methods could facilitate the identification of their roles, enhancing the possibility to fix desirable genotypes and accelerate the breeding rate.
References:
Abdallah NA, Prakash CS, McHughen AG. Genome editing for crop improvement: Challenges and opportunities. GM Crops Food 2015; 6(4):183-205.
Bortesi L, Fischer R. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 2015; 33 (1):41-52.
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