As we are facing a climate change epidemic, alongside population growth and food insecurity, the future of crop production must be carefully considered. Crops may have adapted to certain environmental stresses, however, with weather extremes occurring more frequently, the integration of gene editing technologies may enhance the tolerance, yield, and overall success of crop plants. Failing to increase yields in a sustainable manner will negatively impact our ability to produce large amounts of food in the future. “Sustainable intensification” of agriculture, in which yields are increased without negatively impacting the environment or by bringing in more land, is a target approach, and gene editing may be a successful way to do so. Gene editing involves the modification of an organism’s genetic composition, to make it produce traits advantageous to crop breeding, and therefore, humans. This can be used as a measure to combat food insecurity. Gene modifications can arise using zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), new technologies that are more accurate, affordable, and accessible are needed 1. Genome editing can give rise to targeted modifications in any given crop.
Mutagenesis
Since the discovery during the 1920s that heritable mutations can be induced in plants by radiation or chemical treatments, plant biologists have often taken a mutagenesis approach for gene editing during mutagenesis, the genetic information of a plant is changed, and mutations occur as a result. This can happen accidentally in nature or can be carried out in the lab. Thousands of seeds derived from the crop of interest are mutagenized, and the offspring of the mutagenized population is investigated for possible phenotypes of interest. A single induced mutant can have many traits of interest e.g. drought tolerance and high yield, this is advantageous to transgenic plants where a single gene trait is usually expressed. In term of crop breeding, the rate of spontaneous mutations in transgenic plants is too low to be considered for increasing genetic variation 2.
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Mutagenesis agents include radiation and chemical treatments using Ethyl Methyl Sulfonate. These treatments enhance the rate of mutations, allowing mutant lines to be used for improved crop breeding. During radiation treatment, X-rays and gamma radiation cause bigger chromosome deletions and point mutations. Radiation treatments have shown to be successful. There was record yield increases in rice across Asia in the 1960s due to semi-dwarf varieties of rice (Oryza sativa L.) generated with shorter, stronger stems, as a result of deficiency in gibberellin plant hormone. The mutation is located in the “green revolution gene” 3. Radiation however, is damaging to the surrounding environment.
The chemical mutagen EMS most commonly cause single base substitutions i.e. from G/C to A/T. Chemical mutagens are advantageous as they can give rise to populations of mutants with many mutations, which makes screening for specific mutations in a given population easier 4. An increase in produce shelf life is a widespread advantage derived from chemical mutagenesis. A mutant tomato was developed with a Sletr1-2 gene which gave rise to the increased decay resistance 5.
Limitations
Despite the advantages, limitations to mutagenesis include that plant biologists often “mutate and hope for the best”. 20-100 thousand seeds of a crop must be mutagenized to generate enough offspring to be investigated for desired traits. The mutations that arise from this are completely random, and it’s difficult to detect desired traits that have phenotypes which are not obvious such as certain metabolites and compounds. Often, recessive loss-of-function mutations arise, which decrease the efficiency of the protein encoded by the mutated gene. Pure homozygotes are required to obtain phenotypes when the mutation is recessive. Mutagenesis is a long process that requires labour intensive work, and extensive backcrossing of at least 5 generations is required to remove undesired mutations.
Transgenic approaches
Transgenic techniques can be described as introducing a piece of DNA (such as a gene) from a different species into an organism. This is in contrast to cisgenics, where DNA from a similar species is introduced. Transgenic techniques can be used in crop improvement to introduce new genes into a plant, to generate new, favourable traits for example, resistance to certain diseases. The expression of endogenous genes can also be changed to improve traits for example, overexpression or silencing.
Transgenic plant transformations can be carried out using the TI plasmid of Agrobacterium tumefaciens, a plant pathogenic bacterium that causes tumours in about 60% of dicotyledonous angiosperms and gymnosperms resulting in crown gall disease. This tumour producing property makes it a valuable tool for introducing genes into plants for research and agricultural purposes. The Ti plasmid has the tumour-producing function and is used as part of the genetic equipment to transduce genetic material to plants. Advantages are that Agrobacterium are considered natural genetic engineers and are therefore more widely accepted.
Transfer DNA (T-DNA) moves into the plant cell nucleus, it is flanked by 2 direct 25-bp repeat border sequences. The virulence (vir) genes are required for this movement. The discovery that T-DNA can be inserted into the plant genome raised the possibility that any gene could be transferred into plants. Agrobacterium transfer DNA by chemoattraction and activation of virulance, T-DNA excision, and finally, the movement of T-DNA out of the bacterium.
Applications of A-mediated transfer include the production of GM plants, such as varieties of rice that are resistant to certain diseases and environmental conditions such as drought, facilitating crop improvement. Other applications include basic research, plant transformations allow “in vivo” study of plant genes, transient expression studies, short term expression of T-DNA genes gives faster results than generating transgenic plants. Agrobacterium can also transfer T-DNA into non-plant cells such as animals or fungi.
Limitations include the distance of host range, monocots are not generally infected by Agrobacterium due to wounded monocots not secreting phenoic compounds such as acetosyringone, the inducer of Agrobacterium infection and transcriptional activation of vir operons.
Crispr/cas9
In recent times, plant biologists have delved deeper into the possibilities of gene editing. Although transgenetics, and mutagenesis have shown to be successful, there is a need to be able to precisely alter, replace, or repair an endogenous gene, and to obtain a transgene free genome. CRISPR/CAS9 presents itself as a new and exciting technology for genetic engineering. Clustered Regularly Interspaced Short Palindromic Repeats /CRISPR Associated 9, was first discovered in bacteria and archaea. Its an adaptive immune system found in prokaryotes and can give rise to targeted, heritable mutations, free from transgenes, resulting in non-GM plants. The transgene usually integrates once in the genome of the organism and can be removed by simple selfing and selection of non-transgenic offspring. This gene editing technology displays accuracy, while having a low risk of off target effects when compared to conventional mutagenesis approaches 6.
Other advantages over mutagenesis are that this technology can modify gene regulation through modified cas9 enzyme, can repair genes through homology-directed repair, can add genes at any specific locus on the genome, and can induce mutations in any specific locus on the genome, with the exception of needing a PAM motif next to the guide RNA recognition site, as the sequence of the PAM influences the activity of guide RNA.
CRISPR Applications
In terms of attaining viral disease resistance, two primary actions occur. First, a CRISPR-coding sequence is integrated into the host plant genome. This will target and disrupt the virus genome, with the aim to develop a CRISPR-like immune system in the host genome. Secondly, the induction of a CRISPR-mediated targeted mutation in the host plant genome that will enhance virus resistance. This works when enzyme “cas9” cuts the DNA at a specific location allowing fragments of DNA to then be added or removed. Guide RNA is specific (complementary) to a locus in the genome, ensures the cas9 enzyme cuts at the correct location on the genome. The cell begins to repair the damaged DNA, and plant biologists can use repair machinery to alter genes of interest in the genome. After binding, cas9 acts to cleave DNA, following cleavage, repair machinery will reconnect the separated DNA strands, often creating indels (insertions or deletions). This mutagenizes the target sequence. The cleaved region will be replaced by a homologous section of DNA by the repair machinery, if present in the nucleus.
In a study carried out by P. Ryder et Al. (2017) it was demonstrated that homozygous nulliplex mutants can be directly generated in tetraploid Arabidopsis thaliana plants. this was carried out using CRISPR/Cas9 to generate knockout alleles of the TTG1 gene 7. The mutation of genes in polyploid backgrounds is extremely complex using conventional mutagenesis approaches.
CRISPR/Cas9 genome editing now provides a route to more efficient generation of polyploid mutants for improving understanding of genome dosage effects in plants.
Limitations
CRISPR/Cas9 displays high specificity, compared to mutagenesis, however, off-target regions may also be recognized, and cleaved, resulting in damage. Expected off-target effects occur in genome regions that are similar to the target, but unexpected off-target effects in random regions can also occur. Genome sequence information is necessary for the prediction of expected off-target effects 8. These may lead to the destroying of plasmid DNA or viral nucleic acids, which is beneficial to bacteria and archaea.
Off-target effects can generate undesired mutations at random sites, impacting precise gene modification. Consequences of off-target effects, lack of specificity in targeting, incomplete targeting, and so on, all of which could have negative effects on the crop.
Improved nucleases could potentially increase safety and efficacy of CRISPR/Cas9 use. By reducing off-target effects, the effectiveness of the target gene edit would be increased, and damaging consequences reduced. Greater accuracy would also improve ability to monitor efficacy and safety, over multiple generations [10].
Detecting off-target sites is an ongoing challenge. The T7 endonuclease I assay was developed to identify off-target mutations, however, this assay cannot detect those occurring at Ethical concerns always arise when discussing GMOs. Although genetic modification of plants has been carried out for many years, the idea remains quite alien to many people.
EU regulation currently considers all genetically modified crops or animals as transgenic whether it involves the insertion of foreign DNA or direct genome editing, and therefore subject to regulation and risk assessment 10. Various commentators have requested the reconsideration of regulations as CRISPR/Cas9 and TALENS don’t contain foreign DNA, they should not be considered to be transgenic plants. As the EU is the largest market for agricultural products in the world, other countries are now waiting to see whether they will change the definition of transgenic plants and their regulations before they market edited crops.
Ethical concerns arise when discussing germline editing. CRISPR/Cas9 has potential to be used on embryos to generate resistance to certain diseases, however there are many concerns about potential risk factors for the resulting embryos.
given that CRISPR is cheap, easy to use, and does not require sophisticated equipment or expert knowhow, it has become a popular technology worldwide, which will eventually require international standards for testing genetically edited organisms, releasing them into the environment, and assigning liability for damage. Regulations should set clear requirements for testing the safety and efficacy of edited organisms in carefully controlled environments or contained settings that simulate their natural environments 10.
Releases that may lead to movements of modified organisms with adverse effects on biological diversity or human health.
Future approaches
There is yet another aspect of the genetic editing of microorganisms to consider, as CRISPR could also be used to synthesize and manipulate pathogens, including smallpox, the Spanish flu virus, avian H5N1 flu virus, and SARS. It is not unreasonable to think that, in the wrong hands, CRISPR could be used to make dangerous pathogens even more potent.
Gene editing in seaweeds
Research into genetic transformation systems that are feasible for seaweeds lags far behind that for plants. This is mainly due to the fact that seaweed physiology is still not fully understood, and therefore, most research focuses primarily on seaweed’s physiology and the establishment of cell polarity and multicellularity 11.
Four obstacles to tackle include developing expression constructs suitable for seaweeds cells, transferring the expression constructs into seaweed cells, integration of expression constructs into the genome, and finally, selecting the genetically transformed cells 12. At present, the ZFN genome editing method has been successfully used for targeted mutagenesis in Chlamydomonas reinhardtii 13.
Currently, the most feasible way to establish a system for genetic transformation is to insert foreign genes into the genome by site-specific homologous recombination after introduction of constructs using particle bombardment or microinjection methods and subsequent antibiotic-dependent selection of genetically transformed cells.
Conclusion
As discussed above, gene editing is a promising technology that could massively help overcome the problem of food insecurity, especially as we face environmental changes in the future that could potentially impact crop yield and quality. ZFNs, TALENs, and transgenetics have proven to be successful, and mutagenesis approaches have generated elite varieties of many crops, new technologies are in need of being developed to combat the new challenges we are facing. CRISPR/Cas9 proves to be the gene editing technology of the moment, ticking all the boxes in terms of crop improvement such as being efficient, having an easy to remove Cas9 transgene, the ability to edit and repair genes, and to knock-out mutant generations in a single step, whilst being suitably used for a variety of applications. Using CRISPR/Cas9 for seaweed optimisation is the most prominent future approach.
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