CRISPR in agriculture (Part 48- CRISPR in gene editing and beyond)
Welcome to the 48th part of the multi-part series on applications of CRISPR in gene editing and beyond.
With precise genome editing, CRISPR has the potential to resolve major challenges to crop improvement.
Traditional Selective breeding
Selective plant breeding of crops began thousands of years ago, even when anyone knew about DNA. It is the process of handpicking the best and most desirable traits from different plants and mating them together to create offspring with those desirable traits. The continuous accumulation of spontaneous mutations in the genome is the foundation of genetic variation for species evolution. Mutations cause changes in the DNA of an organism that may result in the natural emergence of plant variants with desired and beneficial traits like higher yield, nutritional content, shelf life, shortened growing seasons, taste less bitter, increased resistance to diseases and pests, can withstand harsh climate conditions, etc. The crops with desired characteristics are selected and bred together. Pollens with the genes for the desired trait from plants of one crop variety are transferred to the flowers of another variety with other desirable traits. From the offspring produced, the farmers again choose those with the best combination of desired traits and breed them together. The process is repeated over many generations to produce the offspring plants that inherit those desirable characteristics. For example, let’s say a farmer wants to grow a crop that produces bigger and sweeter apples. They would start by selecting the apple tree that produces the sweetest apples and then breed it with another tree that produces bigger apples but maybe not as sweet. By cross-breeding these two trees, the farmer can create offspring that inherit the desired size and sweetness traits from both parents.
Our food crops today are, in fact, very different from their wild counterparts. Using selective breeding, farmers have converted wild plants into the crops we rely on today. For example, corn is descended from a wild grass called teosinte, which has small, hard kernels that are not edible. But it has been selectively bred by farmers over thousands of years to produce the much larger, sweeter, and tender corn we enjoy today. Similarly, broccoli, cauliflower, and cabbage all descended from the same wild mustard plant, but through selective breeding, farmers have created distinct varieties with different shapes and flavors. Selective breeding has also been used to create new varieties of other crops, such as wheat, rice, soybeans, and potatoes. Without selective breeding, our modern food supply would look very different, and we would not have access to the wide variety of crops that we have today.
Mutagenesis/Mutation Breeding
In the beginning of the 20th century, plant breeders and researchers learned that with a process called mutagenesis, they could make mutations happen faster than would occur naturally. Physical methods like UV, X-ray, gamma radiation, fast neutron, or chemicals like methyl-N-nitrosourea, hydrogen fluoride, sodium azide, methyl methanesulfonate, or ethyl methanesulfonate can be used to induce mutations in the plant’s DNA. Plant seeds, seedlings, whole plants, or only parts of the plant — pollen, spores, stem of a plant, etc. can be irradiated or given chemical treatment. If the cell’s own repair mechanism does not eliminate the resulting mutations, a heritable mutation is generated. During this process, plant breeders grow, screen, and evaluate each plant from each seed produced. The plants with promising mutations are bred further with the wild plants until the breeders arrive at a substantially improved plant variant that possesses new and desirable characteristics.
More than 3000 desirable crops (including wheat, barley, soybeans, rice, tomatoes, grapefruit, and many fruits) have been developed using radiation mutagenesis in the last 100 years.
Limitations: But there are various limitations of traditional selective breeding and mutation breeding.
(i) Because the mutations induced are random, some may occur in functional genes, making them non-functional. Therefore, a great deal of effort is required to separate undesirable traits from desirable ones, which is sometimes not economically practical. Selecting desirable alleles and their combinations is a long-term process that involves crossing with wild genotypes and cultivating necessary ones for several generations.
(ii) During breeding, DNA from the selected parents may recombine randomly, and desirable traits, such as pest resistance, may get bundled with undesirable traits, such as lower yield or poor quality.
(iii) Additionally, traditional breeding techniques are time-consuming, often take decades to produce new viable crop varieties, and are labor-intensive.
Transgenic plants
The second method for obtaining desired traits lies with genetic engineering and transgenesis. The advantage, compared to induced mutagenesis, is that it allows for obtaining a desired trait through the introduction of a foreign gene (called a transgene) into an organism, which significantly reduces the time required to obtain a genetically modified organism/plant (GMO).
A transgenic crop variety is developed by:
(i) Identifying and isolating a desired gene that confers the trait of interest, for example, a gene responsible for disease or pest resistance, herbicide tolerance, or increased yield.
(ii) The desired gene is cloned into the carrier or vector plasmid.
(iii) The vector containing desired DNA is then introduced into the target plant using various techniques such as Agrobacterium-mediated transformation or gene gun.
(iv) After delivering, the foreign gene integrates into the plant’s DNA and is expressed, resulting in the desired trait.
The modified plants obtained usually contain some DNA sequences from other organisms, sometimes referred to as “foreign DNA” that confer them the desired characteristics. An organism/plant with DNA from another organism is considered transgenic. This technique has widened the range of the modification of plants because transgenes could provide those traits to a recipient plant that could not be obtained by mutagenesis, like the synthesis of pharmaceuticals, insecticides, and other agents in plants. For instance, insect-resistant plants contain genes from the bacteria Bacillus thuringiensis (“Bt genes”) that, when expressed, produce a toxin. The toxin or natural insecticide protects crops from pests but is not harmful to humans. When growing Bt crops, farmers no longer need to use synthetic pesticides, which can be costly, toxic to inhale or handle, and pollute nearby soil and water.
Other examples of transgenic plants are Golden Rice which has a high content of β-carotene, transgenic soybean, etc.
Regulations: Although years of scientific research show that transgenic crops currently available are safe to eat and cause no harm to human health. But they have struggled to gain popularity because of certain concerns:
(i) GMOs may present a certain danger for ecosystems, e. g., how can we be certain that the transgenes in herbicide-resistant plants will not be transferred to weeds by pollens while cross-hybridization?
(ii) The long-term consequences of transgenesis remain unclear; if a transgene gets integrated into different regions of a genome and affects other genes’ expression?
(iii) There is also a risk that a transgenic plant can affect non-target organisms, such as plants possessing BT-toxin genes can kill non-hazardous insects.
(iv) Resistance development: Pests can develop resistance to the toxin produced by transgenic plants, making them less effective over time. This has already been seen in some transgenic crops that produce insecticides, such as Bt cotton, where some pests have developed resistance to the toxin.
(v) Some studies have raised concerns that consuming transgenic crops could have negative health effects on humans or animals, although most studies have found no evidence of harm. For example, some researchers have raised concerns that using antibiotic resistance genes in transgenic crops could lead to the development of antibiotic-resistant bacteria in humans. Another concern is the potential for allergic reactions in people who consume transgenic crops. For example, if a gene from a common allergen is introduced into a crop, it could potentially cause an allergic reaction in people who consume that crop. Despite these concerns, the vast majority of studies have found no evidence of harm from consuming transgenic crops.
To address these concerns, many countries regulate transgenic plants more strictly than non-transgenic plants. Therefore, transgenic plant producers must spend time and money on research to show the public and government that their products are safe for the ecosystem.
(vi) Apart from environmental and health-risk issues, there are several Intellectual property issues. The companies that develop transgenic crops often hold patents on the technology used to create them. They have exclusive rights to produce and distribute the seeds. This means that farmers who want to plant transgenic crops must purchase the seeds from the company that holds the patent, and they may be required to sign a contract that limits their ability to save seeds for future planting or to share the technology with others.
Genome editing of plants by CRISP/Cas9
With the recent advents in genome editing approaches like ZFNs, TALENs, and CRISPR-Cas9 technology, it is possible to precisely generate targeted mutations in the target gene. Whereas, as in the case of conventional mutagenesis, the random and high load of off-target mutations are generated in the plant’s DNA. Thus, if already known which gene is required to be mutated to achieve an improvement, genome editing can massively speed up the process of producing plants with desired traits in the first generation.
Also, genome editing plant breeding technologies differ from transgenic methods in various ways: Genome editing technologies are used for precise genetic manipulation without introducing exogenous DNA such as antibiotic/virus-resistant genes. Thus, eliminating the fear that foreign DNA may be present in the target crop. For example, many viruses need plant proteins to grow, multiply, and spread. Researchers can use genome editing tools to break or change the plant proteins a particular virus likes to take advantage of. As a result, the viruses won’t be able to spread within the edited plants, and thus the plants will become virus resistant. On the other hand, transgenic crop production requires the insertion of foreign DNA to confer virus resistance in the target plant.
Therefore, regulators place less stringent regulations on these non-transgenic, genome-edited plants because they have DNA sequences that could occur naturally in their wild counterparts.
Out of all genome editing tools, CRISPR/Cas9 technique has several advantages over ZFNs and TALENs. It is easy to use, less laborious to make gene editing components, more efficient, cost-effective, has fewer off-target effects, and has the ability to target multiple genes simultaneously, which makes them suitable for editing genomes of numerous polyploid species of crops.
Transgene-free genome editing in plants
The CRISPR-Cas9 system can be used to generate transgene-free genome edited plants. To make the plants completely transgenic-free, the transforming agent is not a vector or plasmid but a ready-to-use complex including Cas9 and a gRNA. Here is a general overview of the steps involved:
(i) Design and synthesize guide RNAs (gRNAs): The first step is to design and synthesize gRNAs that will target the specific DNA sequence to be edited. The gRNAs are typically designed to be specific to the target sequence to avoid off-target effects.
(ii) Delivery of CRISPR-Cas9 components: The CRISPR-Cas9 components, including the Cas9 protein and the gRNA, need to be delivered into the plant cells. This can be done using various methods, such as Agrobacterium-mediated transformation, particle bombardment, or electroporation. After the CRISPR-Cas9 components have been delivered, the Cas9 protein cuts the DNA at the target site, and the plant’s natural repair mechanisms attempt to fix the break. This repair process can result in one of two outcomes: the introduction of small insertions or deletions (INDELs) at the target site or the precise replacement of the target DNA sequence with a new one using the donor template DNA. The donor DNA template can be designed to be similar to the plant’s native DNA sequence, thus avoiding the introduction of foreign DNA.
(iii) Screening for edited plants: Once the plant cells have been transformed with the CRISPR-Cas9 components and donor DNA template, they must be screened to identify those that have undergone successful genome editing. This can be done using various methods, such as PCR amplification and sequencing.
(iv) Regeneration of edited plants: The edited cells are then selected and induced to regenerate into whole plants.
(v) Selection of transgene-free plants: Finally, the plants need to be screened to ensure that they are transgene-free. This can be done using various methods, such as PCR amplification and sequencing, to confirm that there are no foreign DNA sequences in the genome.
By following these steps, it is possible to generate transgene-free genome edited plants using the CRISPR-Cas9 system. This approach has the advantage of avoiding the introduction of foreign DNA into the genome, which can simplify the regulatory process and potentially reduce public concern about genetically modified plants/organisms. However, it is still a relatively new technology and further research is needed to fully optimize and validate the approach.
Applications of CRISPR-Cas9 in crop improvement
CRISPR-Cas9 has revolutionized the field of crop improvement by providing a precise and efficient tool for genome editing. Here are some examples of how CRISPR-Cas9 has been used in crop improvement:
(i) Disease resistance: CRISPR-Cas9 has been used to create crops that are resistant to various diseases caused by viruses, bacteria, and fungi. For example, researchers have used CRISPR-Cas9 to edit the genome of rice plants to make them resistant to bacterial blight, a disease caused by the bacterium Xanthomonas oryzae that can devastate rice crops. CRISPR-Cas9 has also been used to develop wheat that is resistant to powdery mildew, a fungal disease that affects the leaves and stems of plants.
Similarly, CRISPR-Cas9 has been used to create a disease-resistant version of the Cavendish banana, which is the most commonly consumed banana variety worldwide. The Cavendish banana is susceptible to a fungal disease known as Panama disease, which has devastated banana crops in the past. By using CRISPR to edit the banana’s genome, researchers have created a variety that is resistant to Panama disease, offering a promising solution to this ongoing problem.
(ii) Drought and salt tolerance: Drought and high salinity are major challenges to agricultural productivity worldwide, and climate change is expected to exacerbate these challenges in the future. To address this problem, CRISPR-Cas9 can be used to edit genes involved in drought and salt tolerance, leading to crops that can better withstand these harsh conditions. Researchers have used CRISPR-Cas9 to edit the genome of maize, wheat, and rice plants to increase their tolerance to drought, resulting in increased yield in areas with limited water availability. For example, using CRISPR-Cas9 to modify the OsDST gene in rice, researchers have been able to create rice plants that are more tolerant to drought conditions. The gene modification leads to an increase in the accumulation of the stress hormone abscisic acid (ABA) in the plant, which triggers a response that helps the plant to conserve water and survive under drought stress. Similarly, in wheat, researchers used CRISPR-Cas9 to modify a gene called TaHKT1;5, which regulates sodium uptake in the plant. The modification resulted in wheat plants that are better able to tolerate high levels of salt in the soil.
(iii) Improved shelf life: CRISPR-Cas9 has been used to edit genes involved in fruit ripening, leading to crops with improved shelf life. One example of this is the CRISPR-Cas9 modification of the tomato genome to delay fruit ripening and improve its shelf life, resulting in tomatoes that can be transported over longer distances without spoiling. The researchers targeted genes involved in the production of ethylene, a plant hormone that triggers fruit ripening, and were able to create tomato plants that ripen more slowly and stay fresh for longer periods of time.
(iv) Improved crop quality: CRISPR-Cas9 has been used to edit genes involved in crop quality, leading to crops with improved taste, aroma, and nutritional value. For example, researchers have used CRISPR-Cas9 to edit the genome of wheat plants to reduce the levels of gluten, making the wheat more tolerable for people with gluten sensitivity.
(v) Improved yield: CRISPR-Cas9 can be used to edit genes that control key plant growth and development pathways, leading to improved yield. For example, scientists have used CRISPR-Cas9 to increase the number of grains per wheat spike, resulting in higher yield. Another example is the use of CRISPR-Cas9 to modify genes involved in the production of hormones that regulate plant growth and development, such as gibberellins and auxins. By modifying these genes, scientists have been able to create crops that produce more fruit or grain per plant, resulting in increased yield.
(vi) Nutritional enhancement: CRISPR-Cas9 can be used to enhance the nutritional value of crops by editing genes involved in the synthesis and accumulation of nutrients. For example, scientists have used CRISPR-Cas9 to develop rice that produces higher levels of beta-carotene, a precursor of vitamin A, which can help to address vitamin A deficiency and improve public health. Similarly, CRISPR-Cas9 can be used to increase the levels of other important nutrients, such as iron and zinc in crops like wheat, maize, and potatoes. Furthermore, CRISPR-Cas9 can be used to modify the composition of plant oils and fats, making them more healthy for human consumption. For example, scientists have used CRISPR-Cas9 to modify the fatty acid composition of soybean oil, resulting in a healthier oil with lower levels of saturated fats.
(vii) Environmental sustainability: CRISPR-Cas9 can be used to develop crops with reduced environmental impact by editing genes that control plant traits related to nitrogen use efficiency, water use efficiency, and carbon sequestration. For example, scientists have used CRISPR-Cas9 to develop rice that uses nitrogen more efficiently, reducing the need for nitrogen fertilizers. Nitrogen fertilizers are widely used in agriculture to promote plant growth and increase yield, but they can also lead to environmental problems such as nitrogen runoff into waterways and greenhouse gas emissions.
These are just a few examples of the many ways that CRISPR-Cas9 is being used to improve crops. As the technology continues to advance, we will likely see even more applications of genome editing in crop improvement.
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