Authors: Sushma Rani1 and Kiran B. Gaikwad2
1Senior Research Fellow, Division of Genetics, Indian Agricultural Research Institute, Pusa campus, New Delhi-110012.
2Scientist, Division of Genetics, Indian Agricultural Research Institute, Pusa campus, New Delhi-110012.
One of the most significant recent technologies rapidly emerging for genetic improvement of microbes, plants, animals and its specific cells and developing embryos is genome engineering. Genome editing or Targeted Genome Modifications (TGM) is an emerging technology and swiftly gaining researcher’s interest for crop improvement as it can change the specific chromosomal sequences with great precision. The editing of the chromosomal region may comprise an insertion, substitution or deletion of at least one nucleotide and the edited gene sequence should translate modified gene product with altered/new function. Genome editing requires molecular scissors and accessory tools which comprise mega-nucleases, site-specific recombinases (SSRs), recombination enzymes, synthetic oligonucleotides, and engineered viruses. DNA repair systems such as homologous recombination (HR) pathway and non-homologous end joining (NHEJ) pathway of the cell are exploited the most for genome editing including gene knockout (disruption), knock-in (insertion), and allelic exchange. The HR DNA repair system for genome editing was the most initial approach exploited.
The idea of genome editing came into existence the early 1990s when bacterial type II restriction enzymes were first used to target a specific chromosomal region. In 2007, Mario Capecchi, Sir Martin Evans, and Olivier Smithies won the Nobel Prize for Physiology or Medicine for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells. Scientists are adopting this advanced technique to investigate the genome functionally and the further can be attempted to develop crops resistant to biotic and abiotic stresses, enhancing nutritional value etc. (Voytas, 2013). In past few years, reverse genetics has been exploited for disclosing gene functions and annotating DNA sequences. This approach basically includes knock-in and knockout using various approaches such as i) transposon-mediated modification, ii) site-specific recombinase and iii) RNA interference (RNAi) (Tierney and Lamour 2005, Townsend et al., 2009). However, gene targeting has proved to be the most straightforward among the different forms of genetic modification techniques, for the exploration of gene function in vivo. Genetic modification using transposon will directly or indirectly influence the expression level of the gene targeted/induced generated by the random positional insertion of genes, while RNAi has transitory knockdown response, unstable off-target impact and too much background noise (Martin and Caplen 2007; Chen et al., 2014). However, there are several key considerations for gene editing; confirmatory genotyping strategies, off-target site analysis, modification expression and contamination (Chen et al., 2014)
Why is genome engineering required?
Developing Genetically Modified (GM) crops is expensive and biosafety studies necessary to meet the regulatory requirements substantially add to this cost (Lusser et al., 2012; Lucht 2015). In past decade, generations of GM crops have so far relied on introducing indigeneous/foreign DNA sequences to the genome in a random location. One of the important concern of this approach was the position of an introgressed gene in the host genome; as it may alter or inactivate the functionality of other important genes nearby.
Genome editing might prove to be more acceptable to the public than plants genetically engineered (GM crops/Transgenics) with foreign DNA in their genomes. Genome editing is also most accepted as it occurs as a natural process without artificial genetic engineering and it enables modification in a specific area of the DNA, thereby promoting the precision of the correct editing or insertion and offers perfect reproducibility. Various tools are available for TGM which can effectively modify the gene(s) as per the scientific objectives.
Tools for genome editing
Genome-editing methods are initiated by a site-specific double-stranded break (DSB) in a chromosomal region of DNA. A range of molecular scissors for inducing DSBs at specific sites of a genome are available with genome editors. Such molecular scissors comprise engineered, site-specific nucleases (SSNs), such as meganucleases (MNs), also known as homing nucleases (HNs), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided nuclease (RGN) systems. The most widely used RGN being the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated system 9 (CRISPR/Cas9), and DNA-guided nuclease (DGN) system, which introduces DNA DSBs and consequently triggers DNA repair pathways.
- Genome Editing Mediated by Site-Specific Recombinase (SSRs) SSRs are the specific class of enzymes which basically promotes DSBs. Site-specific recombinase (SSR) is an effective genetic engineering strategy which leads to a permanent alteration in the target DNA of the genome. Genome editing has also been done through Transposons and Group II intron retrotransposons and site-specific recombinase systems, such as Cre/loxP and Flp/FRT (Allen and Weeks 2009; Wang et al., 2011). SSRs are an effective aid for editing of the specific target region of genomes and also for activating or de-activating and altering the gene expression levels in numerous organisms (Wang et al., 2011). The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. In addition, recombinases have also been demonstrated to be capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Wang et al. provided alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes (Allen and Weeks 2009, Wang et al., 2011). Some engineered plant viruses (e.g., RNA virus, tobacco rattle virus (TRV), and single-stranded DNA (ssDNA) viruses called Gemini viruses) have been used as genome-editing devices that act as delivery vehicles of SSNs.
- Genome Editing Mediated by Site-Specific Nucleases (SSNs)
- Meganuclease-Based Engineering
c.1) Zinc Finger Nuclease-Based Engineering
A zinc-finger nuclease (ZFN) has a modular structure and occurs in almost all transcription factors that are composed of two domains: a DNA-binding zinc-finger protein (ZFP) domain and the nuclease domain derived from the FokI restriction enzyme (Kim and Kim 2014). Each ZF has recognition site at C-terminal and recognizes a 3-bp small DNA sequence (Petolino 2015, 2016). Four to six zinc fingers (ZFs) are used to generate a single ZFN subunit that binds to the DNA sequences of 9–18 bp. Target sequences of ZFN pairs are typically 18–36 bp in length, excluding spacers. Importantly, the DNA-binding specificities of ZFs can be altered by mutagenesis, which is a key feature of constructing a programmable nuclease. However, the major drawback of this approach is its complexity, off-target consequences and several optimizations needed which is very costly.
c.2) TALEN System
A new class of engineered nucleases, TALENs, was first reported by Christian et al. (2010)for increasing efficiency, safety and accessibility of genome editing. TALENs are detected in plant pathogenic bacterium Xanthomonas and are important virulence factors that act as transcriptional activators in the plant cell nucleus, where they directly bind to specific DNA. TALENs exhibit a DNA-binding domain that binds to numerous plant promoters while translating its protein into the host plant (Boch et al., 2009). The TALENs are artificial endonucleases created by fusing the DNA-binding domain of TALE protein and the DNA cleavage domain of the FokI restriction enzyme which can recognize 13-bp or longer target DNA (Cěrmák et al., 2011). The big obstacle in applying TALEN system is in constructing the vector with suitable monomers for binding the target DNA in the genome.
c.3) CRISPR/CAS Genome Editing System
Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have proven to be one of the most versatile tools for genome editing.. CRISPRs are genetic elements which provide immunity to bacteria against viruses. The protection mechanism includes 3 main stages: adaptation, transcription, and interference. At the adaptation stage, foreign DNA when entered bacterial cells, a small fragment is incorporated into the CRISPR locus of the host genomic region in specialized repeat structures separated from each other by short palindromic repeats and therefore they received the name CRISPR. The Cas genes (CRISPR associated), are localized very close to the CRISPR cassette express protein and have helicase and nuclease activity (Haft et al., 2005). At transcription level, the entire CRISPR locus is transcribed into a long pre-crRNA (poly-spacer precursor crRNA) and produce short crRNAs (CRISPR RNA) of 39–45 nucleotides containing one spacer sequence. The ability to provide defense against invading genetic elements seems to render CRISPR/ Cas systems particularly desirable in hostile environments (Godde and Bickerton 2006). Cas9 generates cleavage at both strands of the target DNA and binding specificity is defined solely by the chimeric crRNA:tracrRNA molecule. CRISPR has been adapted to create RNA directed genome engineering tools by introducing some modifications, including codon CRISPR has been adapted to create genome engineering tools directed by RNA by introducing some modifications, including codon optimization for the Cas9 nuclease for adequate transcription in higher eukaryotic cells, fused to nuclear localization signals (NLS). Besides this, a single chimeric guide sgRNA (crRNA tracrRNA duplex) is constructed under the control of promoter type III instead of expressing 2 non-coding RNAs (tracrRNA and pre-crRNA)
Within this RNA, a stretch of 20 bases is complementary to the respective target site. The CRISPR/Cas system is especially attractive because of its very simple design process. One needs to only insert the desired DNA oligonucleotide into a vector construct for target site selection, as specificity is solely defined by base complementarity to the guide RNA (gRNA). The Cas9 protein does not require any re-engineering and has worked well for all of the target sites. By contrast, the CRISPR/Cas effector module, which is involved in the the crRNAs maturation as well as in its target recognition and cleavage, shows a far greater diverse versatility (Mohanraju et al., 2016).
Crop Improvement by gene editing
Genome editing can targets modifications in the genome very efficiently, easily and precisely. This technology can help to improve important food crops against biotic and abiotic stresses making them ready for mitigating climate change. The presence of novel alleles in nature either in wild relatives or emerged from directed mutagenesis allows the breeder to improve crops by traditional breeding, genetic engineering or genome editing. In addition, mutation breeding is regularly employed in development of new alleles, but due to their low frequency and random nature of this event they could affect many non-targeted alleles, therefore, they must be subjected to substantial screening and backcrosses. Genome editing, however, targets a specific position in the genome, so less screening or backcrossing is required. In addition multiple alleles could be modified simultaneously using CRISPR/Cas9 system. Although genome editing for crop improvement is in its infancy, important traits in major crops have been subjected to modifications using this technology.
Ishii and Araki (2016) have weighed the favorable and unfavorable factors of genome-editing technologies from consumers’ perspectives. The favorable factors of genome-editing include: (a) gene addition via HDR would be easy to understand because of its similarity to transgenesis, (b) transgene flow never occurs except for crops in which a transgene was added via HDR, (c) food safety can be improved much more superiorly as compared to GM crops because of the lack of transgenes in crops modified via NHEJ, in addition to precise gene modification, and (d) the regulatory framework was established in Argentina and is being established in New Zealand.
Tobacco plants containing mutations in the AHAS gene were produced which expressed high levels of herbicide tolerance (Kochevenko and Willmitzer, 2004). Chimeric RNA/DNA oligonucleotide was delivered to tobacco mesophyll cells either by protoplast electroporation or by particle bombardment. Okuzaki and Toriyama (2004) delivered RNA/DNA chimeric oligonucleotides to rice callus cells by microprojectile bombardment targeting the P171A mutation in ALS. Combinations of mutant FAD2 and FAD3 genes in soybean were used to produce high oleic acid and low linolenic acid soybean oil allowing the accumulation of monounsaturated fats and reduce the level of linolenic acid in seeds, producing healthier and improved shelflife oil (Pham et al., 2012). In rice, the protein produced by the OsSWEET14 gene contributes to pathogen survival and virulence of the bacterial pathogen, Xanthomonas oryzae (Li et al., 2012). Genome editing using TALEN was used to generate mutation in the target-binding site in the promoter region of OsSWEET14, thereby inhibiting the transcription of the protein and simultaneously reducing the pathogen’s virulence. Beetham et al. (2014) and Sauer et al. (2016) reported use of various chemistries known to induce DSB, in combination with delivery of the oligonucleotide template, to dramatically increase the efficiency of ODM in Arabidopsis and Linum usitatissimum (Flax). Herbicide-tolerant canola plants have also been produced using ODM (Gocal, 2012; Gocal, 2015). This was achieved by propagation of aseptically grown in vitro plants from seed or from microspore-derived embryos.
Genome-editing technology, in general, and crop improvement, in particular, if the regulatory authority authorizes the commercialization of GEOs are less burdensome, less time-consuming, and formed after detailed deliberations with all concerned parties so that they can be applied globally. Above all, the CRISPR/Cas patent dispute needs to be settled as early as possible so that the potential of this excellent technology is fully exploited.
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