Going back to the basics - what is genome engineering and CRISPR/Cas?
To begin with, genome engineering or genome editing is a way of making specific changes to the DNA of a cell or organism. One way that scientists use genome editing is to investigate different diseases that affect humans, such as cancer. They edit the genomes of organisms because animals share many of the same genes as humans. Similarly, gene editing can also be used for plants or any crop species to enhance certain traits or remove undesirable genes, making this technique extremely useful for agriculture.
There are many editing techniques such as transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFN), and CRISPR/Cas. For this article, we will look at the attempts to use CRISPR/Cas in agriculture to solve the global hunger crisis.
The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is essentially an RNA-mediated adaptive immune system that occurs naturally in bacteria and archaea. CRISPR is a specialised region of DNA with two distinct characteristics, namely the presence of nucleotide repeats and spacers.
Repeated sequences of nucleotides (i.e. the building blocks of DNA) are distributed throughout a CRISPR region. On the other hand, spacers are bits of DNA that are interspersed among these repeated sequences. In the case of bacteria, the spacers are taken from viruses that previously attacked the organism, and they serve as a bank of memories which enables bacteria to recognise the viruses and fight off future attacks.
In order to successfully cleave the foreign DNA, the CRISPR/Cas system requires crRNA, tracrRNA, Cas9, and PAM sequences. Here’s a brief overview of their respective roles:
CRISPR RNA (crRNA)
Once a spacer is incorporated and the virus attacks again, a portion of the CRISPR is transcribed and processed into CRISPR RNA, or "crRNA." The nucleotide sequence of the CRISPR acts as a template to produce a complementary sequence of single-stranded RNA. Each crRNA consists of a nucleotide repeat and a spacer portion.
Trans-activating crRNA (tracrRNA)
The protein typically binds to two RNA molecules: crRNA and another called tracrRNA. The two then guide Cas9 to the target site where it will make its cut. This expanse of DNA is complementary to a 20-nucleotide stretch of the crRNA.
Cas9
The Cas9 protein is an enzyme that cuts foreign DNA.
Protospacer Adjacent Motifs (PAMs)
There is a built-in safety mechanism, which ensures that Cas9 doesn't just cut anywhere in a genome. Short DNA sequences known as PAMs ("protospacer adjacent motifs") serve as tags and sit adjacent to the target DNA sequence. If the Cas9 complex doesn't see a PAM next to its target DNA sequence, it won't cut.
Using two separate regions, or "domains" on its structure, Cas9 cuts both strands of the DNA double helix, making what is known as a "double-stranded break”. The application of CRISPR in mammalian cells was simplified by fusing tracrRNA and crRNA to create single-guide RNA (gRNA). As a result, genome editing can be done in any sequence by defining the targeting sequence of gRNA (Figure 1).
Figure 1. The CRISPR/Cas System. This figure illustrates guide RNA (gRNA), Cas9, PAM sequence (highlighted in orange colour) and the DNA strands with matching genomic target sequence. Gene editing can lead to one of these two possible results, namely gene disruption and gene insertion. Figure created on BioRender.
Why is genome engineering relevant to agriculture?
With the rise of climate change and population issues, food security is becoming one of the biggest concerns. Given that the global population is expected to reach over 10 billion by 2100, this raises the question about the sustainability and sufficiency of food resources to meet the demand.
Previously, crops were modified through conventional breeding. However, with the development of biotechnology, genome editing was made possible using site-specific nucleases such as transcription activator-like effector nucleases (TALEN) and zinc-finger nucleases (ZFN), and these effectively replaced the slow process of breeding.
Nonetheless, the recent discovery of CRISPR/Cas9 system from bacteria revolutionised the genome editing system as a result of its robustness. Beyond enabling a convenient, simple and effective targeted genome editing, CRISPR can allow for a more efficient gene editing and has other potential applications such as the ability to regulate gene expression or modification of chromosomes (which will be discussed in the future outlook section).
From Table 1, it seems clear that CRISPR/Cas9 provides a huge advantage over TALEN and ZFN.
Table 1. A comparison of features between TALEN, ZFN, and CRISPR/Cas9
Application of CRISPR/Cas in plant genome editing
CRISPR-based plant genome editing starts with the identification of a target site. Using bioinformatics tools such as ‘CRISPR Design’, we can essentially construct our very own sgRNA. The sgRNA, along with Cas9, gets cloned into either a single or binary vector. A vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material, typically using a bacterial plasmid. While a single vector uses a single plasmid, a binary vector uses two plasmids to produce a genetically modified organism. Following that, the incorporation of nuclear localisation signals (which is a signal that helps the protein to be translocated to the target organelle) into the Cas protein enables successful integration of the CRISPR/Cas9 system in plant nuclei.
There are many ways to transform the vectors into plant cells, however, Agrobacterium-mediated transformation is mostly used due to high efficiency and stability. Following callus (i.e. the culturing of dedifferentiated plant cells) culture and regeneration, the mutants are screened using methods such as qPCR, which helps the researchers rapidly amplify DNA. Lastly, the whole plant genome is sequenced to detect targeted mutations and off-target sites via Sanger or next-generation sequencing, which are techniques used to help researchers determine the full nucleotide sequence of the DNA sample. Finally, the phenotype of the plant is characterised. These transgenic plants with the desired trait can then be self-pollinated for phenotypic analysis of plants in the subsequent generations (Figure 2).
Figure 2. Workflow of plant GE using the CRISPR/Cas system. The process starts with the target site identification and gene selection. Then, gRNA is synthesised according to the target site gene and sgRNA and Cas9 are cloned in vectors under suitable promoters. After delivery of the vector into the plant cell, the grown callus with the transformed vectors is screened to detect target mutations. Lastly, the phenotypes of the plants are characterised. Figure created on BioRender.
CRISPR/Cas9 has been used to edit the genes of major crops such as rice (Oryza sativa), maize (Zea Mays), soybean (Glycine max), wheat (Triticum aestivuum), tomato (Solanum lycopersicum), potato (Solanum tuberosum), cucumber (Cucumis sativus), and barley (Hordeuum vulgare). In the context of agriculture, crop gene editing is important as they serve as a critical staple food. Particularly, rice and wheat provide approximately 20% of the calories and over 25% of the protein consumed by humans.
Case studies
Application in metabolic engineering
Metabolic engineering involves the modification of the metabolic composition of the cell. Effective strategies include over-expressing or suppressing key regulators in biosynthetic pathways. Zhang et al. (2020) utilised CRISPR/Cas9 platform to suppress competing metabolic pathways of isoflavone synthesis in soya bean. Isoflavones play a critical role in plant-environment interactions and are beneficial for human health, as it could, for example, reduce the risk of developing cancer. By knocking out genes that encode key enzymes, the mutants showed twice the isoflavone content compared to the wild type. The results also showed that increasing isoflavones can improve resistance to soya bean mosaic virus (SMV), one of the most harmful diseases of soya beans, and these two traits were shown to be.
Application in plant synthetic biology
Meanwhile, metabolic engineering can also be utilised in the field of synthetic biology.
Synthetic biology involves the synthesis of complex systems to produce a complete metabolic pathway. One example of an on-going study is the C4 rice project, which aims to make photosynthesis efficiency in the C3 metabolic pathway in rice similar to that of C4 plants such as maize. Both pathways involve the use of carbon dioxide (CO2) fixing enzyme rubisco.
However, C4 rubisco has a faster carboxylation rate and therefore can increase rice yield up to 50%. Candidate genes are those encoding for rubisco and for the C4 differentiation process. The current work is focused on identifying the regulatory genes in maize and understanding the biochemical pathway.
With further advancements, CRISPR may be used in this project through multiplex gene replacement. Similarly, seeing that genes can be altered not only in the nucleus but also in the chloroplast, further studies about mitochondrial and chloroplast DNA may elucidate their involvement in trait improvement. In the near future, it may even be possible to assemble desirable genes to create a “super-crop”.
Future outlook
Beyond simple gene editing such as gene disruption and insertion, the CRISPR/Cas system has other potential applications such as transcription regulation, and epigenomic editing, and gene drive.
Transcription regulation involves CRISPR Interference (CRISPRi) and CRISPR Activation (CRISPRa) which results in transcription repression and activation respectively. Transcription can also be regulated by epigenetic modifications by using methyl- or acetyltransferases. Methyltransferases are enzymes that help suppress DNA transcription by adding methyl groups onto DNA, while acetyltransferases are enzymes that help activates transcription by adding acetyl groups onto DNA. These applications require the use of modified cas9 nuclease, which is known as ‘dead’ cas9 (dCas9). dCas9 contains mutations so they lack nuclease activity but does not hinder target binding. The new developments in cas9 can enhance specificity, adaptability, reversibility and allow gene editing without causing DNA breaks.
CRISPR shows great potential to be used as a tool for gene drive. “Gene drive” is a genetic engineering technique designed to help the genes spread through a population at propagation rates much higher than normal. This technique requires a plasmid containing the Cas9 gene and a gRNA, that can insert itself into the cleavage site of the. Once inserted, the gene can propagate itself and be inherited onto the majority of progeny. So far, this technique has been used for pest control such as malaria. As demonstrations of a functional synthetic drive in a plant population are currently lacking, theoretical and experimental approaches are needed to understand and predict the applicability of such a technique in the field of agriculture.
Safety issues and regulations
Despite several potential applications of CRISPR in agriculture, the progress mainly depends on the regulatory environment. There is an on-going debate globally on whether or not CRISPR-produced food should be considered as genetically modified organisms (GMOs).
GMOs require regulatory approval before release and the process involves the assessment of risks to human health and the environment. Hence, many issues regarding GMOs arise due to the fact that this type of genetic engineering involves the insertion of transgenic material into the organism. However, CRISPR eliminates the issue of GMOs because CRISPR engineers the organism’s genome instead of inserting a foreign genetic material. At the same time, CRISPR still requires the delivery of a bacterial vector, so this topic remains controversial.
If CRISPR would be considered as a safe alternative to the production of GMO foods, it may be useful for countries where GMOs are not allowed. Figure 4 highlights the EU countries (including the U.K) where GMO production is approved and banned.
Recently, in 2018, a European court stated that CRISPR-altered crops are GMOs that need strict regulation. By contrast, the U.S Department of Agriculture (USDA) exempts them from GMOs. The matter of safety is perhaps the major drawback for plant genome engineering as this technology cannot be put into practice without approval. Therefore, this divisive issue needs to be resolved on how CRISPR-engineered foods would be regulated between different countries. It also seems that the definition of GMOs needs to be clearer.
Figure 4. Map of Europe showing EU countries where GMO production is approved and banned. The countries where GMO cultivation and sale are allowed (green), the countries where such actions are banned (red), and the countries that do not belong to the EU (grey).
Why should we consider the application of CRISPR in agriculture?
Although safety issues are one of the main limitations, the application of CRISPR in agriculture has multiple advantages over other fields. For example, the biomedical application requires the consideration of in vivo delivery and ethical issues. The process is also time-consuming because multiple clinical testing needs to be done to see the success rate. Similarly, its application in machine learning seems to be hampered by the variation among different algorithms. However, plant genome engineering is a relatively straightforward and convenient method, thus it eliminates most of the limitations that such fields face.
Due to the simplicity, versatility and efficiency of CRISPR/Cas, a lot of progress has been made to understand the role of CRISPR in the field of agriculture. Clearly, this technique can be used not only in simple genome editing but also beyond this field, offering various ways to study and modify the genome. One example is its application in drug discovery, as it makes it easier to create cellular and whole-animal model systems that precisely mimic diseases. This enables scientists to more accurately verify the safety and efficacy of drugs.
Henceforth, in the near future, CRISPR can accelerate plant biology research, ultimately advancing current crop traits and even generating new crops. Therefore, with further developments, the CRISPR toolkit would play a critical role in addressing the issues of food security in the face of climate change and population growth.
Author
Yunji Choi
BSc Biochemistry
Imperial College London
Disclaimer: All figures created using BioRender are intended solely for educational purposes and not for profit.
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