Dr Rhitu Rai- Scientist, National Research Centre on Plant Biotechnology (NRCPB), LBS building, New Delhi-110012
Dr P K Dash- Senior Scientist, National Research Centre on Plant Biotechnology (NRCPB), LBS building, New Delhi-110012
Genome engineering is the field of research in reverse genetics dedicated to developing heritable changes in the genome in a sequence specific manner or site directed manipulation of a genome. This ability to specifically add or delete genetic information has enabled us with an unmatched level of precision in studies of gene function in vivo. Historically, the field of plant breeding has been utilizing natural allelic variation in the germplasm or randomly inducing mutations by chemical mutagens like ethylmethanesulphonate (EMS) or gamma rays. But this involves large-scale screens of mutagenized plant populations to identify those rare plants with the desired phenotype followed by considerable efforts to identify the sequence modification responsible for change in phenotype. Other reverse genetic tools like TILLING (for Targeting Induced Local Lesions in Genomes) transfer DNA and (T-DNA) insertional mutagenesis have made it easier to link the gene to the phenotype it is responsible for. TILLING involves traditional chemical mutagenesis followed by high-throughput screening for point mutations whereas T-DNA is a method of insertional mutagenesis involving the a segment of the Ti plasmid of Agrobacterium tumefaciens known as T-DNA that carries genes to transform the plant cell. But here again, creating the genetic variation is largely a matter of chance. The recent advanced approaches like RNA interference (RNAi) and artificial microRNAs for gene disruption are more targeted and hence sequence information is a prerequisite. Both the techniques have provided immense breakthroughs in functional genomics but are limited by incomplete generation of knockouts as they have often been found to be leaky.
The recent evolution of targeted mutagenesis and DNA insertion techniques based on tailor-made site-directed nucleases (SDNs) provides opportunities for better utilization of rich genomic resources available in plants for reverse genetics. These techniques utilize the
These techniques are based on combination of following two important factors:
• DNA binding domain provided by modular nature of Zinc finger motifs, Transcription activator like effector motifs
• Catalytic domain provided by FokI nuclease
These two fused together gives the ability to introduce double strand breaks (DSBs) at specific locations within the genome. This is followed by harnessing the inherent repair mechanism for the DNA double strand break (DSB) repair. Two primary mechanisms are used to correct DSBs: homologous recombination (HR), in which DNA templates bearing sequence similarity to the break site are used to introduce sequence changes to the target locus, and nonhomologous end joining (NHEJ), in which the broken chromosomes are rejoined, often imprecisely, thereby introducing nucleotide changes at the break site.
Zinc Finger Nucleases (ZFNs)
ZFNs were the first sequence-specific nucleases engineered to recognize and cleave novel chromosomal sites. Each Zinc finger recognizes three nucleotides and three or four zinc fingers comprise one array which functions in pairs. A total of 18-24 bp is recognized at a given target site which is a sufficient length to be unique in genome.
Transcription Activator-Like Effector Nucleases (TALENs)
TALENs (TALE nucleases) are fusion proteins of transcription activator-like effectors (TALEs) and the DNA cleavage domain of FokI nuclease. TALes are proteins produced by plant bacterial pathogens of the genus Xanthomonas. During infection, Xanthomonas delivers to the plant cell that bind to specific plant gene promoters and activate expression. This, in turn, leads to increased pathogen virulence. DNA binding by TAL effectors is carried out by the protein's central domain, which comprises 13-28 copies of a typically 34- amino-acid repeat. Each repeat recognizes a single base, and there is a one-to-one correspondence between the repeats in the protein and the bases in the DNA they bind. The amino acid sequence of each repeat is highly conserved, with the exception of the so-called repeat variable di-residues (RVDs) at amino acid positions 12 and 13. Each 34-amino-acid repeat folds into a hairpin-like structure in which the RVDs are positioned at the tip. The 12th amino acid reaches back to stabilize the hairpin, whereas the 13th amino acid makes a base-specific contact in the major groove of DNA. The most common RVDs--the amino acid residues NI, HD, NG, and NN--bind to adenosine, cytosine, thymine, and either guanine or adenosine, respectively. This cipher can be exploited to engineer TALEs or DNA binding domains that specifically target to the preselected DNA sequences in rice. Designer TALEs (dTALEs) can be used for targeted gene activation and thus gene function analysis. TALEN technology has been exploited to edit the specific rice genes to thwart the virulence strategy of Xoo and, thereby, engineer heritable genome modifications for blight resistance (. Multiple TALENs are custom-engineered to precisely edit the TALE binding elements within the promoters of the disease susceptibility genes. The resultant promoter modifications in rice plants result in loss of inducibility of susceptibility genes by the cognate TALEs and concomitantly loss of susceptibility (or gain of resistance) to pathogen. The TALEN gene constructs can be genetically segregated out from the modified plants. The results have demonstrated the feasibility of using TALENs for targeted editing for crop improvement and also raise the prospect of producing non-genetically modified organisms.
Though plant breeding and genetically modified crops have made valuable contributions to sustainable agriculture, targeted genome engineering holds a great promise to elevating crop yields.
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