Genome editing has revolutionized the field of genetics, providing scientists with unprecedented tools to modify DNA sequences with precision. While the majority of research has focused on diploid organisms, the significance of genome editing in polyploids is an emerging frontier that holds immense promise. Polyploidy, the condition of having multiple sets of chromosomes, is widespread in plants, and some animals, fungi, and bacteria. By applying genome editing we can destroy the binding sites of different types of bacteria and fugus in a plant and can increase food production. This article delves into the unique challenges and opportunities associated with genome editing in polyploid organisms.

What Is Polyploidy?

Polyploidy is the term used to indicate a plant’s genomic nature of having multiple sets of chromosomes. However, polyploids, characterized by their possession of more than two sets of homologous chromosomes, exhibit enhanced genetic diversity and often display increased robustness and adaptability. In plants, polyploidy is a common phenomenon, with many crops, such as wheat, cotton, and potatoes, being polyploid. Despite their prevalence, manipulating the genetic makeup of polyploids has traditionally posed challenges due to the complexity of multiple copies of genes and interactions.

What Are The Challenges of Genome Editing In Polyploids?

Allelic Redundancy

In diploid organisms, a single copy of a gene exists on each chromosome. In contrast, polyploids carry multiple copies of each gene, leading to allelic redundancy(more than one gene is acting for a single biological process and deactivation of one gene can have little/no effect in the organism). Editing a specific gene in a polyploid organism requires targeting all copies, making it a more intricate process.

Homeolog Interaction

Polyploid genomes often contain homeologs, which are similar but not identical copies of genes derived from different parental genomes. The interaction between homeologs complicates the precise targeting of genetic modifications, as changes made to one copy may not necessarily affect the others.

Off-Target Effects – What Is off-target Effects In Genetics?

Off-target effects means, a gene editing tool (CRISPR-Cas9) is making changes on a non-targeted gene sequence and because of the change we observe adverse effect.

Polyploid genomes may exhibit higher chances of off-target effects due to the presence of multiple copies of genes. Ensuring the specificity of genome editing tools in such complex genetic landscapes is a considerable challenge.

What Are The Genome Editing Techniques of Polyploids?

Genome editing in polyploids presents unique challenges due to the presence of multiple copies of homologous genes and complex interactions among them. However, several advanced techniques have been developed to overcome these challenges and enable precise modifications in the genomes of polyploid organisms.

CRISPR/Cas Systems

The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) system is a revolutionary genome editing tool that has been adapted for use in various organisms, including polyploids. CRISPR technology enables the targeted modification of specific DNA sequences by guiding the Cas9 or other Cas proteins to the desired genomic loci. In polyploids, multiplex genome editing can be employed to simultaneously target multiple copies of genes.

Base Editing

Base editing is a precise genome editing technique that allows the conversion of one DNA base pair into another without causing double-strand breaks. This method is particularly useful in polyploids, where precise modifications are crucial to avoid unintended effects on multiple gene copies. Base editing offers a higher degree of accuracy and reduced off-target effects compared to traditional CRISPR/Cas systems.

Prime Editing

Prime editing is another advanced technique that offers high precision in genome editing. It allows the direct rewriting of genomic DNA by introducing new genetic information without requiring a donor template. This technique is beneficial in polyploids, as it facilitates the modification of specific gene copies without disturbing the overall genomic structure.

RNA-Guided Epigenetic Editing

Epigenetic modifications play a crucial role in regulating gene expression. RNA-guided epigenetic editing involves the use of modified Cas proteins to introduce specific epigenetic changes, such as DNA methylation or histone modifications. This technique can be applied in polyploids to modulate gene expression without altering the underlying DNA sequence.

Multiplex Automated Genome Engineering (MAGE)

MAGE is a technique that allows the simultaneous modification of multiple genomic loci. In polyploids, MAGE can be employed to edit several gene copies simultaneously, providing a more efficient way to introduce complex modifications. This method is particularly useful for engineering polyploid organisms with improved traits or novel functionalities.

Synthetic Biology Approaches

Synthetic biology techniques involve the design and construction of novel biological systems. In polyploids, synthetic biology approaches can be utilized to engineer entire pathways or networks of genes to achieve specific goals. These approaches may involve the assembly of synthetic gene circuits or the introduction of entirely new metabolic pathways.

Homoeologous Recombination

Homoeologous recombination takes advantage of the natural recombination events that occur between homoeologous chromosomes in polyploid organisms. By using CRISPR/Cas systems to induce double-strand breaks at specific loci, researchers can stimulate homoeologous recombination, promoting the exchange of genetic material between homologous chromosomes.

Meganucleases (MNs) Technique

Meganucleases (MNs), also known as homing endonucleases, are DNA-cleaving enzymes with high sequence specificity. These enzymes can be used for targeted genetic editing in polyploids, though their use may present some challenges due to the presence of multiple copies of homologous genes. It has a large cleavage site to process homoeologous recombination in cells. The double stranded DNA break occurred by MNs can be reversed by using non-homologous end joining (NHEJ) or homology-directed repair (HDR). There are five families of MNs:

• GIY-YIG
• HNH
• His-Cys box
• PD-(D/E) XK
• LAGLIDADG

MNs can be a good choice to handle target-site polymorphism.

Zinc-finger nucleases (ZFNs) Technique

Genome editing in polyploids using Zinc-Finger Nucleases (ZFNs) involves targeted modification of specific DNA sequences by inducing double-strand breaks at desired locations. Although polyploidy introduces challenges, ZFNs provide a powerful tool for precise genetic modifications. It has three specific sites:

• DNA-binding domain (Cys2–His2)
• DNA recognition module
• DNA-cleavage domain

It can make high mutation on target site.

Transcription activator-like effector nucleases (TALENs) Technique

Genome editing in polyploids using Transcription Activator-Like Effector Nucleases (TALENs) involves the targeted introduction of double-strand breaks (DSBs) in specific genomic loci, followed by the cell’s natural repair mechanisms to achieve desired genetic modifications.

At a glance we can say that, ‘DNA binding domain including catalytic domain of the Fok I endonuclease’ makes TALENs. It has the largest target site compared to MNs. With this technique we can target every sequence in an interval of 10bp but the problem is we need to engineer a new chimeric protein for every target. As the size if TALENs is so large it’s not efficient for plant cells. However, scientist developed new technology called ‘de novo engineered transcription activator-like effector (dTALEs)‘ for plant cells.

Despite these advanced techniques, challenges in achieving efficient and specific genome editing in polyploids persist. Optimizing delivery methods, minimizing off-target effects, and enhancing the scalability of these techniques are ongoing areas of research. As technology continues to advance, the potential for harnessing the benefits of genome editing in polyploids will undoubtedly expand, opening new avenues for improving crop traits, biodiversity conservation, and understanding the complexities of polyploid genomes.

How To do Gene Editing in Polyploids with Meganucleases (MNs)?

Meganucleases (MNs), also known as homing endonucleases, are DNA-cleaving enzymes with high sequence specificity. These enzymes can be used for targeted genetic editing in polyploids, though their use may present some challenges due to the presence of multiple copies of homologous genes. Below is a general guide on how to perform genetic editing in polyploids using Meganucleases:

1. Designing Meganucleases for Polyploid Genomes:
   • Identify the target gene or genes within the polyploid genome that you want to edit.
   • Design Meganucleases with high specificity for the target sequences. Consider the challenges of polyploidy, such as allelic redundancy and homeolog interactions, during the design phase.
2. Constructing Meganuclease Expression Vectors:
   • Clone the Meganuclease gene into an expression vector under the control of a suitable promoter.
   • Include other elements like terminators, selection markers, and any necessary regulatory sequences.
   • Ensure that the expression vector is compatible with the host organism’s cellular machinery.
3. Transformation of Polyploid Cells:
   • Choose an efficient transformation method for introducing the Meganuclease expression vector into the target cells of the polyploid organism. Methods may include Agrobacterium-mediated transformation for plants or protoplast transformation for fungi.
   • Optimize the transformation protocol to enhance the efficiency of delivering the Meganuclease into the cells.
4. Selection of Edited Cells:
   • Introduce a selectable marker, such as a gene for antibiotic resistance, along with the Meganuclease to aid in the selection of successfully edited cells.
   • Allow time for the transformed cells to proliferate and express the selectable marker.
5. Screening for Targeted Genetic Edits:
   • Develop a screening strategy to identify cells with the desired genetic modifications.
   • Employ molecular techniques, such as PCR or DNA sequencing, to verify the presence of the intended edits in the target gene or genes.
   • Account for potential challenges like allelic redundancy and the need to differentiate between homoeologs.
6. Off-Target Analysis:
   • Assess potential off-target effects by analyzing other genomic loci with sequences similar to the target site.
   • Minimize off-target effects by using carefully designed Meganucleases and optimizing experimental conditions.
7. Propagation of Edited Cells:
   • Once edited cells are identified and confirmed, propagate these cells to generate a population of organisms with the desired genetic modifications.
   • Monitor and characterize the edited traits to ensure stability and heritability.
8. Validation and Characterization:
   • Conduct thorough validation experiments to confirm the stability and heritability of the edited traits.
   • Characterize the phenotypic changes resulting from the genetic edits in the polyploid organisms.
9. Iterative Optimization:
   • If necessary, iterate the process, optimizing the Meganuclease design or experimental conditions to enhance efficiency and specificity.

How To do Gene Editing in Polyploids with Zinc-finger nucleases (ZFNs)?

Here is a step-by-step guide on how to perform genome editing in polyploids using ZFNs:

1. Target Selection:
   • Identify the target gene or genes within the polyploid genome that you want to edit.
   • Design ZFNs with high specificity for the target sequences. Consider the challenges of polyploidy, such as allelic redundancy and homeolog interactions, during the design phase.
2. Constructing ZFN Expression Vectors:
   • Design and construct ZFNs by fusing zinc-finger DNA-binding domains to the FokI endonuclease domain. The resulting ZFNs should recognize and bind to the target sequences.
   • Clone the ZFNs into an expression vector under the control of a suitable promoter.
   • Include other elements like terminators, selection markers, and any necessary regulatory sequences.
   • Ensure that the expression vector is compatible with the host organism’s cellular machinery.
3. Cell Transformation:
   • Choose an efficient method for introducing the ZFN expression vector into the target cells of the polyploid organism. Methods may include Agrobacterium-mediated transformation for plants or electroporation for certain cell types.
   • Optimize the transformation protocol to enhance the efficiency of delivering the ZFNs into the cells.
4. Selection of Edited Cells:
   • Introduce a selectable marker, such as a gene for antibiotic resistance, along with the ZFNs to aid in the selection of successfully edited cells.
   • Allow time for the transformed cells to proliferate and express the selectable marker.
5. Screening for Targeted Genetic Edits:
   • Develop a screening strategy to identify cells with the desired genetic modifications.
   • Employ molecular techniques, such as PCR or DNA sequencing, to verify the presence of the intended edits in the target gene or genes.
   • Account for potential challenges like allelic redundancy and the need to differentiate between homoeologs.
6. Off-Target Analysis:
   • Assess potential off-target effects by analyzing other genomic loci with sequences similar to the target site.
   • Minimize off-target effects by using carefully designed ZFNs and optimizing experimental conditions.
7. Propagation of Edited Cells:
   • Once edited cells are identified and confirmed, propagate these cells to generate a population of organisms with the desired genetic modifications.
   • Monitor and characterize the edited traits to ensure stability and heritability.
8. Validation and Characterization:
   • Conduct thorough validation experiments to confirm the stability and heritability of the edited traits.
   • Characterize the phenotypic changes resulting from the genetic edits in the polyploid organisms.
9. Iterative Optimization:
   • If necessary, iterate the process, optimizing the ZFN design or experimental conditions to enhance efficiency and specificity.

How To do Gene Editing in Polyploids with Transcription activator-like effector nucleases (TALENs)?