Revolutionizing Genetics: The Emergence and Impact of Gene Editing Technologies
Exploring the Revolution of Gene Editing: From CRISPR to DNA Nanoparticles to Gene Drives
Abstract:
In recent years, gene editing has emerged as a rapidly developing field that is transforming the way genetic research is conducted. The discovery of the CRISPR/Cas9 system has revolutionized the field, making it more accessible and efficient. It has enabled scientists to target and modify specific genes with unprecedented precision and ease, opening up new avenues of research and a wide range of applications in medicine, nanotechnology, and molecular engineering. Recent advances have greatly enhanced the capabilities of gene editing systems, making the field uniquely positioned to shape millions of lives. The impact of gene editing has already been demonstrated, as seen in the lipid-nanoparticle delivery system used in the Pfizer-Biontech Covid-19 vaccine.
Outline of Paper
- Gene Editing in a Nutshell
2. New CRISPR-based modalities
2.1. Base editors
2.2. Prime editors
2.3. RNA-targeting Cas13 effectors
3. Gene-Editing Mechanisms
3.1. ZFNs
3.2. TALENs
3.3. CRISPR
4. Germline editing.
4.1. Yamanaka Factors
4.2. iPSC
4.3. Epigenetic alterations
5. Gene drive technologies
5.1. Molecular mechanisms
5.2. Gene Propagation
6. DNA Nanoparticles
6.1. Structure DNA Nanotechnology (SDNs)
6.2. Dynamic DNA Nanotechnology (DDNs)
6.3. Nanomedicine
7. Homology-Directed Repair
7.1. Classical double-strand break repair (DSBR)
7.2. Synthesis-dependent strand-annealing (SDSA)
7.3. Break-induced repair (BIR)
9. Conclusion
1. Gene Editing in a Nutshell
Gene editing is a field of biotechnology that has made significant advancements since its inception. In the 1970s, scientists conducted the first recombinant DNA experiment, which allowed for the transfer of genetic material between different organisms. This groundbreaking experiment laid the foundation for modern genetic engineering and gene editing. In the 1980s, techniques were developed for introducing specific mutations in genes, including site-directed mutagenesis. This allowed researchers to study the function of specific genes and develop new therapies for genetic diseases.
In the 1990s, the CRISPR-Cas9 system was discovered in bacteria. The CRISPR-Cas9 system is a powerful gene editing tool that allows scientists to cut DNA at specific locations and introduce changes, deletions, or insertions. While initially used in bacteria, this system was eventually adapted for use in gene editing in other organisms, including humans. In the 2000s, RNA interference (RNAi) was developed as a tool for gene knockdown and gene expression regulation. RNAi is a natural process that cells use to regulate gene expression, and it has since been harnessed as a powerful tool for gene editing and drug discovery.
In the 2010s, the CRISPR-Cas9 system was refined and widely adopted as a powerful gene editing tool, thanks to Jennifer A. Doudna and Emmanuelle Charpentier. The system has numerous applications in research, medicine, and agriculture. In research, it has been used to study the function of genes and develop new therapies for genetic diseases. In medicine, it has the potential to treat a wide range of genetic disorders, including sickle cell anemia, cystic fibrosis, and Huntington’s disease. In agriculture, gene editing has been used to create genetically modified crops with improved yields, resistance to pests and diseases, and other desirable traits.
2. New CRISPR-based Modalities
The development of new CRISPR-based modalities has been driven by the need for more precise and versatile gene editing tools. While the original CRISPR-Cas9 system was a groundbreaking discovery that revolutionized the field of genetic engineering, it has some limitations that make it less effective for certain applications. For example, a study published in the journal Nature Biotechnology in 2017 found that the Cas9 protein can sometimes cause large deletions or insertions of genetic material near the site where it cuts the DNA. This can lead to unintended changes in the function of the gene or even the loss of important genetic information.
The new CRISPR-based modalities have been developed to address some of these limitations and provide more precise and flexible tools for genetic engineering.
2.1. Base Editors
Base editors are a type of CRISPR-based gene editing tool that allow for targeted changes to individual bases in the DNA sequence without cutting the DNA. Unlike traditional CRISPR-Cas9 gene editing, which relies on the Cas9 enzyme to cut the DNA at specific locations, base editors use various enzyme to chemically modify the DNA base at a specific location.
The two most commonly used types of base editors are cytosine base editors (CBEs) and adenine base editors (ABEs). CBEs convert cytosine bases to uracil, which is then converted to a thymine base during the cell’s natural DNA repair mechanisms. ABEs, on the other hand, convert adenine bases to inosine, which is then converted to a guanine base during DNA repair.
Base editors have a number of unique applications in gene editing, particularly in cases where precise changes to individual bases in the DNA sequence are required. For example, base editors have been used to correct genetic mutations associated with cystic fibrosis and hereditary deafness, as well as to create new genetic variations that can be used in crop breeding.
One of the main advantages of base editors is that they can be more precise and efficient than traditional CRISPR-Cas9 gene editing. Because they do not cut the DNA, there is less risk of unintended mutations or other unintended effects. In addition, base editors can be used to create specific point mutations without introducing new genetic material, which can be useful in cases where the introduction of new genes is undesirable. However, base editors also have some limitations, including a limited range of target sites.
2.2. Prime Editors
Prime editors are a type of CRISPR-based gene editing tool that allow for precise and efficient changes to the DNA sequence without cutting the DNA. They were developed to overcome some of the limitations of traditional CRISPR-Cas9 gene editing, which can result in unintended mutations and other off-target effects.
Prime editors consist of a modified Cas9 enzyme, called a nickase, and an engineered reverse transcriptase enzyme. The nickase cuts one strand of the DNA at a specific location, while the reverse transcriptase uses RNA templates to create new DNA sequences that are then inserted into the nicked strand of the DNA.
One of the main advantages of prime editors is their ability to make precise changes to the DNA sequence without introducing new genetic material or cutting the DNA. They can be used to make targeted point mutations, insertions, and deletions, as well as to correct genetic mutations that cause disease. Another advantage of prime editors is their ability to edit DNA sequences that are difficult to target with traditional CRISPR-Cas9 gene editing. For example, prime editors can be used to make changes in regions of the DNA that are inaccessible to the Cas9 enzyme, such as in repetitive sequences or in areas with high levels of chromatin. Prime editing systems such as PASTE also offer the capability to edit kilobases of sequences, orders of magnitude more capable than the traditional CRISPR/Cas9 system which could edit a dozen bases as best.
2.3. RNA-targeting Cas13 effectors
CRISPR-Cas13 proteins are RNA-guided RNA nucleases, therefore targetting RNA molecules rather than DNA. They were first discovered in bacteria and were later adapted for use in human cells. Cas13 effectors consist of a Cas13 protein, which is guided to a specific RNA sequence by a guide RNA molecule. Once the Cas13 protein binds to the target RNA, it activates its RNA-cleaving activity, which destroys the RNA molecule.
Despite being RNA nucleases, Cas13 effectors are capable of inhibiting the infection of phage DNA. Moreover, these characteristics make Cas13 effectors ideal candidates for broad-spectrum phage editing, applicable in phage therapies to address superbugs. Upon target RNA binding, Cas13 unleashes general, non-specific RNA degradation that arrests growth of the virocell to block infection progression, thereby limiting infection of neighboring cells.
One of the unique applications of RNA-targeting Cas13 effectors is their ability to be used as a diagnostic tool for detecting specific RNA sequences. This is achieved by coupling the Cas13 protein to a reporter molecule that produces a detectable signal when the RNA molecule is cleaved. This technique, known as SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing), has shown promise for detecting viral infections and genetic diseases.
3. Gene-Editing Mechanisms
Gene editing mechanisms refer to the techniques used to alter the DNA sequence of an organism. There are several methods of gene editing, including CRISPR/Cas9, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). CRISPR/Cas9 is currently the most widely used gene editing tool, which uses a guide RNA to target specific sequences of DNA and a Cas9 protein to cut the DNA. Once the DNA is cut, the cell’s natural repair mechanisms can be used to introduce new genetic material, delete existing genetic material, or make precise changes to the DNA sequence.
3.1. ZFNs
Zinc finger nucleases (ZFNs) are a type of genetically engineered DNA-binding proteins that enable precise and targeted genomic editing through the induction of double-strand breaks at specific user-defined genomic loci. These double-strand breaks serve as triggers for the natural DNA-repair machinery of the cell, including homologous recombination and non-homologous end joining (NHEJ). By leveraging established protocols and techniques, these DNA repair pathways can be directed to achieve site-specific mutagenesis, such as the deletion, integration, or modification of target genes in cell lines.
ZFNs have been used for a variety of applications in medicine, including gene therapy, disease modelling, and drug discovery. One of the most promising applications of ZFNs is in gene therapy for genetic diseases. ZFNs can be used to correct genetic mutations by introducing a specific DNA sequence at the site of the mutation, thereby restoring normal gene function. For example, ZFNs have been used to correct mutations in the gene that causes severe combined immunodeficiency (SCID), a rare genetic disorder that severely compromises the immune system. ZFNs have also been used to correct mutations in the gene that causes hemophilia B, a blood-clotting disorder.
ZFNs have also been used to create disease models for a variety of genetic disorders, including Huntington’s disease, cystic fibrosis, and muscular dystrophy. By introducing specific mutations into cells, researchers can study the underlying mechanisms of these diseases and develop new treatments. ZFNs have also been used in drug discovery to create cell lines with specific mutations for screening potential drugs. Overall, ZFNs have shown great promise in medicine, but their use has been somewhat limited by their complexity and difficulty of use compared to newer gene-editing technologies such as CRISPR-Cas9.
3.2. TALENs
TALENs (Transcription Activator-Like Effector Nucleases) are a type of engineered DNA-binding proteins that are used to create targeted double-stranded breaks in the genome. They are comprised of two functional domains: the DNA-binding domain, which is derived from transcription activator-like effectors (TALEs), and the FokI nuclease domain, which is used to cleave the DNA. The DNA-binding domain of TALENs is designed to specifically recognize and bind to a user-specified DNA sequence, and once bound, the FokI domain cleaves the DNA at the target site, leading to the induction of site-specific mutations.
The applications of TALENs in medicine are numerous. One of the most promising applications is in the development of gene therapies for inherited diseases caused by single-gene mutations. TALENs can be used to correct these mutations by inducing targeted DNA breaks and stimulating the cell’s DNA repair machinery to introduce a corrected version of the gene. Additionally, TALENs can be used to create animal models of human diseases by introducing specific mutations into the genome. This is particularly useful for studying the mechanisms of disease and for testing potential therapies. TALENs have also been used to engineer cells for use in cell-based therapies, such as creating immune cells that are resistant to HIV infection.
3.3. CRISPR
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) works by using a protein called Cas9, which acts as a pair of molecular scissors. The Cas9 protein can be programmed to target and cut specific DNA sequences using RNA molecules that are complementary to the target DNA. The cell’s natural DNA repair mechanisms then fix the cut, either by inserting or deleting DNA sequences, which can change the function of the gene.
The potential applications of CRISPR in medicine are vast and include:
- Cancer Treatment: In a study published in Science Translational Medicine in 2016, researchers used CRISPR to modify T cells to recognize and attack cancer cells. The modified T cells were then used to treat patients with metastatic lung cancer, and some patients showed significant improvements in their condition.
- Huntington’s Disease: Researchers at the University of California, San Francisco, used CRISPR to correct the gene responsible for Huntington’s disease in a mouse model of the disease. The study, published in the journal Nature in 2019, showed that the CRISPR-edited mice had improved motor function and a reduced accumulation of the protein that causes the disease.
- Duchenne Muscular Dystrophy: In a study published in the journal Science in 2018, researchers used CRISPR to correct the genetic mutation responsible for Duchenne muscular dystrophy in a mouse model of the disease. The CRISPR-edited mice had improved muscle function and a longer lifespan than untreated mice.
4. Germline editing.
Germline editing is a type of genetic modification that involves making permanent changes to the DNA in reproductive cells, such as sperm or eggs. This means that any changes made through germline editing would be passed on to future generations, affecting not only the individual being edited but also their offspring and descendants.
4.1. Yamanaka Factors
Kyoto University researcher Shinya Yamanaka accomplished the conversion of somatic cells into induced pluripotent stem cells, which are akin to cells found in embryos. This means that any non-reproductive cell from a person’s body can be transformed into an embryonic-like cell that can divide without limit, multiply, and develop into a specific cell type like nerve cells or blood cells.
Yamanaka’s team analyzed 24 genes that were highly expressed and specific to pluripotent cells. They integrated retroviruses carrying these genes into adult somatic cells, such as skin cells, to express them. After a period of incubation, they introduced a marker gene that was only activated in pluripotent cells. The team repeated this process to narrow down the cocktail of genes to four reprogramming factors: Klf4, Sox2, Oct4, and Myc. These genes are transcription factors, meaning they can bind to DNA and regulate the expression of other genes. When all four of these genes are introduced into a mature cell, they can reset the cell to a pluripotent state, which means it has the potential to differentiate into any type of cell in the body.
4.2. iPSC
This process of reprogramming mature cells into pluripotent stem cells is called induced pluripotent stem cell (iPSC) technology. iPSCs have significant implications for regenerative medicine because they can be used to generate cells and tissues for transplantation without the risk of rejection by the immune system.
The applications of the Yamanaka factors in medicine are vast and include the following:
- Tissue regeneration: iPSCs can be used to generate cells and tissues that can be used for transplantation to treat diseases such as Parkinson’s, diabetes, and heart disease.
- Disease modeling: iPSCs can be generated from patients with genetic diseases and used to study the mechanisms of disease and test potential treatments.
- Drug discovery: iPSCs can be used to create cell-based models for drug screening and development, reducing the need for animal testing.
- Personalized medicine: iPSCs can be generated from individual patients and used to create personalized treatments for a variety of conditions.
While iPSC technology has shown great potential in preclinical studies, there are still many technical challenges to overcome before it can be used in clinical settings. One major concern is the risk of tumor formation, as the Yamanaka factors can also induce cell proliferation. Nevertheless, the Yamanaka factors represent a major breakthrough in stem cell research and have the potential to transform the field of regenerative medicine.
4.3 Epigenetic alterations
Epigenetic alterations refer to chemical changes that can occur in DNA or associated proteins that influence gene expression without changing the underlying DNA sequence. These alterations can include DNA methylation, histone modification, and RNA-associated silencing mechanisms. DNA methylation involves the addition of a methyl group. A well-studied example of such an alteration is the methylation of DNA cytosine to form 5-methylcytosine. This usually occurs in the DNA sequence CG, changing the DNA at the CG site from CG to 5-mCG. Methylation of cytosines in CG sites in promoter regions of genes can reduce or silence gene expression.
Histone modifications, such as acetylation or methylation, can alter the structure of chromatin, which can in turn can also influence gene expression. Acetylation of histone proteins can neutralize the positive charge on lysine residues within histones, reducing the electrostatic attraction between histones and negatively charged DNA. This can lead to a more open chromatin structure, allowing transcription factors and RNA polymerase to access and transcribe the DNA sequence more easily, resulting in increased gene expression.
Methylation of histone proteins can have different effects depending on the specific lysine residue that is methylated. For example, methylation of lysine 4 on histone H3 (H3K4me) is often associated with active transcription and increased gene expression, while methylation of lysine 9 on histone H3 (H3K9me) is often associated with gene silencing and decreased gene expression.
RNA-associated silencing mechanisms, such as microRNAs, can also prevent the translation of mRNA into proteins. Epigenetic alterations can be influenced by various environmental factors, such as diet, stress, and exposure to toxins, and can contribute to the development of various diseases and disorders. Understanding epigenetic alterations is important in the study of gene regulation and the development of potential therapies for various health conditions.
5. Gene drive technologies (GDTs)
Gene drive technologies are a method of genetic engineering that can promote the spread of a specific set of genes throughout a population by modifying the likelihood that a particular allele will be passed down to offspring, rather than the usual 50% chance governed by Mendelian inheritance. This process can occur naturally or be engineered, and has the potential to be a powerful tool for selectively modifying populations or species. The method can utilize gene addition, deletion, disruption, or modification.
5.1. Molecular mechanisms
At a molecular level, an endonuclease gene drive functions by cutting a chromosome at a specific site that does not encode the drive, causing the cell to repair the damage by copying the drive sequence onto the damaged chromosome, resulting in two copies of the drive sequence. This method is based on genome editing techniques and exploits the fact that double-strand breaks are usually repaired through homologous recombination, which uses a template, rather than non-homologous end joining. To accomplish this behaviour, endonuclease gene drives consist of two nested elements: a homing endonuclease or RNA-guided endonuclease (such as Cas9 or Cas12a) and its guide RNA that cuts the target sequence in recipient cells, and a template sequence used by the DNA repair machinery after the target sequence is cut. To ensure that the gene drive can spread through the population, the repair template must contain at least the endonuclease sequence. Since the template must be used to repair a double-strand break at the cutting site, its sides are homologous to the sequences adjacent to the cutting site in the host genome. By targeting the gene drive to a coding gene sequence, the gene can be inactivated, and new functions can be introduced by adding extra sequences to the gene drive. As a result, the gene drive insertion will occur in each organism that inherits one copy of the modification and one copy of the wild-type gene. If the gene drive is already present in the egg cell, all the gametes of the individual will carry the gene drive, unlike in normal genes where only 50% of gametes would contain the gene.
5.2. Gene Propagation
The proposed applications of gene drives involve eradicating disease-carrying insects, controlling invasive species, and eliminating herbicide or pesticide resistance. However, as with any powerful technique, gene drives can have unintended consequences or be misused. For instance, a gene drive intended for a local population may spread across an entire species, and eradicating invasive species may have consequences for the species as a whole, even in their native habitat. Gene drives can be constructed using naturally occurring selfish genetic elements, which induce similar segregation distortion in the wild. Most gene drives have been developed in insects, such as mosquitoes, to control insect-borne diseases.
“Singapore’s National Environment Agency breeds millions of mosquitoes in an effort to reduce the mosquito population in the city-state, injecting them with the bacterium Wolbachia. When scientists infect the local mosquito population with Wolbachia, the insects can no longer transmit dengue fever readily to humans. Additionally, under some circumstances, the bacterium can interfere with mosquitoes’ ability to reproduce.” — Source
A gene drive introduced in a single individual typically requires dozens of generations to substantially affect a population, as it can never more than double in frequency with each generation. However, releasing drive-containing organisms in sufficient numbers can have a more immediate impact; for instance, by introducing it in every thousandth individual, it takes only 12–15 generations to be present in all individuals. The fixation and speed of a gene drive’s spread in a population depend on its effect on individual fitness, the rate of allele conversion, and the population structure. Population genetics predicts that in a well-mixed population with realistic allele conversion frequencies (around 90%), gene drives can become fixed for a selection coefficient smaller than 0.3; in other words, gene drives can spread modifications as long as reproductive success is not reduced by more than 30%. This contrasts with normal genes, which can only spread across large populations if they increase fitness.
Until recently, the assumption had been that gene drives could only be engineered in sexually reproducing organisms because the strategy requires the simultaneous presence of an unmodified and a gene drive allele in the same cell nucleus. This excludes bacteria and viruses, as they do not reproduce sexually. However, during a viral infection, viruses can accumulate multiple genome copies in infected cells, and cells are often co-infected by multiple virions. Recombination between viral genomes is a well-known and widespread source of diversity for many viruses, particularly herpesviruses, which are nuclear-replicating DNA viruses with large double-stranded DNA genomes that frequently undergo homologous recombination during their replication cycle. These properties have enabled the design of a gene drive strategy that does not involve sexual reproduction. Instead, it relies on the co-infection of a cell by a naturally occurring and engineered virus. When co-infection occurs, the unmodified genome is cut and repaired by homologous recombination, producing new gene-drive viruses that can gradually replace the naturally occurring population. In cell culture experiments, it was shown that a viral gene drive can spread into the viral population and significantly reduce the virus’s infectivity, which could lead to new therapeutic strategies against herpes viruses.
6. DNA Nanoparticles
DNA nanotechnology involves the construction of artificial nucleic acid structures for various technological purposes. Unlike their natural function as carriers of genetic information in living organisms, nucleic acids are utilized as non-biological engineering materials for nanotechnology. Scientists have developed both static structures, such as 2D and 3D crystal lattices, nanotubes, polyhedra, and arbitrary shapes, and functional devices, including molecular machines and DNA computers. The field is now being used to solve basic science problems in structural biology and biophysics, as well as to explore potential applications in nanomedicine and molecular-scale electronics.
Nucleic acids’ strict base pairing rules allow for the rational design of complementary sequences, which can selectively assemble into complex target structures with precisely controlled nanoscale features. Tile-based, DNA origami, and strand displacement methods are some of the techniques used to construct these structures. Although the field is commonly referred to as DNA nanotechnology, it also includes other types of nucleic acids, leading to the alternative name nucleic acid nanotechnology.
Properties of nanoparticles:
Nucleic acids possess unique properties that make them ideal for use in nanotechnology, which involves the study of materials and devices on a scale below 100 nanometers. In DNA nanotechnology, molecular self-assembly takes place bottom-up, with molecular components spontaneously organizing into stable structures based on the physical and chemical properties of the selected components. Synthetic strands of nucleic acids, such as DNA, are commonly used in this field, as they are well-suited for nanoscale construction. The simple base pairing rules that govern the binding between two nucleic acid strands make it easy to control the assembly of nucleic acid structures through nucleic acid design. This is in contrast to other materials used in nanotechnology, such as proteins and nanoparticles, which lack the capability for specific assembly on their own.
The structure of a nucleic acid molecule is determined by the sequence of nucleotides, which are distinguished by the nucleobase they contain. In DNA, the four bases present are adenine (A), cytosine (C), guanine (G), and thymine (T). Complementary sequences of nucleotides will bind to each other to form a double helix, with A only binding to T, and C only to G. This property allows for the rational design of base sequences so that they will selectively assemble into the desired conformation. The patterns of binding in a system of strands are determined by the sequences of bases, which can be easily controlled through nucleic acid design. While DNA is the most commonly used nucleic acid in nanotechnology, other nucleic acids such as RNA and peptide nucleic acid (PNA) have also been used to construct structures.
6.1. Structural DNA Nanotechnology (SDNs)
SDN, or Structural DNA Nanotechnology, is a field that concentrates on producing and analyzing nucleic acid materials and complexes that reach an equilibrium endpoint during assembly. The DNA double helix structure has a stable, well-defined three-dimensional shape, making it feasible to anticipate, simulate, and plan complex structures that use nucleic acids. Scientists have successfully created various structures, such as two- and three-dimensional lattices, and discrete and periodic structures, using this method.
Lattice Structures
The process of creating extended lattices involves the assembly of small nucleic acid complexes with sticky ends into larger two-dimensional periodic lattices, which display a specific tessellated pattern of the individual molecular tiles. One of the earliest examples of this approach used double-crossover (DX) complexes as the basic tiles, which were designed to combine into periodic two-dimensional flat sheets with a rigid, crystal-like structure. Other motifs have also been used to create two-dimensional arrays, including the Holliday junction rhombus lattice and various DX-based arrays using a double-cohesion scheme. These tile-based periodic lattices are shown in the top two images at the right.
In addition, two-dimensional arrays can be made to exhibit aperiodic structures whose assembly implements a specific algorithm, showcasing one form of DNA computing. The sticky end sequences of the DX tiles can be chosen to act as Wang tiles, allowing them to perform computation. A DX array whose assembly encodes an XOR operation has been demonstrated, enabling the DNA array to implement a cellular automaton that generates a fractal known as the Sierpinski gasket. Another system acts as a binary counter, displaying a representation of increasing binary numbers as it grows. These results illustrate the potential for computation to be incorporated into the assembly of DNA arrays. The third image at the right shows an example of an aperiodic two-dimensional lattice that assembles into a fractal pattern.
Discrete structures
The DNA origami method is commonly used to create nanostructures with complex and irregular shapes. This technique involves using a natural virus strand as a “scaffold” and computationally designed short “staple” strands to fold it into the desired shape. Unlike other DNA nanotechnology methods, this technique does not require high strand purity or accurate stoichiometry, and is easy to design since the base sequence is predetermined by the scaffold strand sequence. Initially, this method was used to create two-dimensional shapes, such as a smiley face, a map of the Western Hemisphere, and the Mona Lisa painting. Solid three-dimensional structures can be made by arranging parallel DNA helices in a honeycomb pattern, while two-dimensional structures can be folded into a hollow three-dimensional shape like a cardboard box. These structures can be programmed to respond to a stimulus and open, releasing a molecular cargo, making them potentially useful as programmable molecular cages.
Templated assembly
The process of templated assembly involves incorporating heteroelements, such as proteins, metallic nanoparticles, quantum dots, amines, and fullerenes, into nucleic acid structures. This enables the creation of materials and devices with a wider range of functionalities than is achievable with nucleic acids alone. The approach uses the self-assembly of nucleic acid structures to direct the assembly of nanoparticles, controlling their position and orientation. Covalent attachment methods are commonly employed, utilizing oligonucleotides with functional groups like amide or thiol to bind the heteroelements. This technique has been successfully used to arrange gold nanoparticles and Streptavidin protein molecules into specific patterns. Another example is nucleic acid metallization methods, which involve replacing the nucleic acid with a metal that assumes the shape of the original nucleic acid structure.
6.2 Dynamic DNA nanotechnology
The goal of dynamic DNA nanotechnology is to create nucleic acid systems that have pre-designed dynamic functionalities that are related to their overall structures, such as mechanical motion and computation. While there is some overlap with structural DNA nanotechnology, as structures can be formed through annealing and then reconfigured dynamically or can be made to form dynamically from the start.
Strand Displacement Cascades
Cascade reactions involving strand displacement can be utilized for both structural and computational purposes. In a single strand displacement reaction, a new sequence is exposed in response to the presence of a trigger strand. Several of these reactions can be connected into a cascade, where the output sequence of one reaction can trigger another strand displacement reaction elsewhere. This allows for the construction of complex chemical reaction networks with the ability to process information and perform computations. The formation of new base pairs and entropy gain from disassembly reactions makes these cascades energetically favorable. Moreover, strand displacement cascades can operate isothermally, without the need for a thermal annealing step, which is required in traditional nucleic acid assembly processes. The initiator species can also support catalytic function, where a single initiator molecule can trigger the entire reaction.
Strand displacement complexes can be used to create molecular logic gates, which can perform complex computations. Unlike electronic computers that use electric current as inputs and outputs, molecular computers use the concentration of specific chemical species as signals. In nucleic acid strand displacement circuits, the signal is the presence of nucleic acid strands that are released or consumed by binding and unbinding events to other strands in displacement complexes. This technique can be employed to construct logic gates such as AND, OR, and NOT gates. Recently, a four-bit circuit was demonstrated to compute the square root of integers 0–15, using a system of gates that contained 130 DNA strands.
6.3. Nanomedicine
Nanomedicine refers to the use of nanotechnology in the field of medicine, encompassing a wide range of applications such as nanomaterials and biological devices, as well as nanoelectronic biosensors.
Drug Delivery
Nanotechnology offers the potential for targeted drug delivery to specific cells using nanoparticles. This approach allows for the active pharmaceutical agent to be deposited in the affected area only, minimizing overall drug consumption and side effects. Nanoengineered devices such as Lipid/DNA nanoparticles can potentially achieve this through molecular targeting. Nanoscale medical technologies are less invasive, faster, and more sensitive than typical drug delivery methods, and can be implanted inside the body.
7. Homology-directed repair & Non-homologous end joining (NHEJ)
Homology-directed repair (HDR) is a type of DNA repair mechanism that uses a homologous DNA sequence to repair a damaged strand of DNA. In HDR, a template with a complementary sequence is used to guide the repair of the broken or damaged DNA strand. This process is typically used by cells during the S and G2 phases of the cell cycle to repair DNA double-strand breaks. HDR is an important mechanism for maintaining genomic stability and preventing mutations and genetic diseases. It is also used in genome editing techniques such as CRISPR-Cas9 to introduce precise changes to DNA sequences in cells. There are various types of HDR, notably DSBR, SDSA and BIR.
Non-homologous end joining (NHEJ) on the other hand is a process of DNA repair that occurs in cells. It is a mechanism by which broken ends of double-stranded DNA molecules are rejoined. This process can occur in any stage of the cell cycle and does not require a homologous template for repair.
NHEJ involves the binding of Ku proteins to the broken DNA ends, which then recruits other proteins such as DNA-PKcs, XRCC4, and Ligase IV to form a complex that aligns and ligates the DNA ends back together. This can sometimes result in deletions or insertions at the site of the repair due to the imprecise nature of the joining.
NHEJ is an important mechanism for repairing DNA damage caused by ionizing radiation, reactive oxygen species, and other genotoxic agents that induce DNA double-strand breaks. It is also involved in V(D)J recombination during immune system development, where it plays a critical role in the generation of antibody diversity.-
7.1. Double-strand break repair (DSBR)
In the conventional double-strand break repair (DSBR) pathway, repair synthesis is initiated by the invasion of the 3’ ends into an undamaged homologous template, creating a primer for DNA repair. This process leads to the formation of four-stranded branched structures known as double Holliday junctions (dHJs), as the invasive strand elongates and synthesizes DNA from the second DSB end. To resolve the individual HJs, cleavage occurs either on the crossing or non-crossing strand. Horizontal cleavage occurs at the purple arrows, while vertical cleavage occurs at the orange arrows. When resolved dissimilarly (e.g., one junction is cleaved on the crossing strand and the other on the non-crossing strand), a crossover event occurs. In contrast, if both HJs are resolved in the same manner, this leads to a non-crossover event.
7.2. Synthesis-dependent strand-annealing (SDSA)
SDSA is a repair mechanism that results in non-crossover events only, which conserves all newly synthesized sequences in the original damaged DNA molecule. Unlike in DSBR, the newly synthesized portion of the invasive strand is displaced from the template and rejoins the processed end of the non-invading strand at the other DSB end after strand invasion and D-loop formation. The 3' end of the non-invasive strand is elongated and sealed to fill the gap, effectively concluding the SDSA process.
7.3. Break-induced repair (BIR)
BIR involves the repair of one-ended DSBs where there is only one invading strand available for repair. Primary synthesis is initiated from a template using this single invasive strand, which is then followed by lagging strand synthesis to fill in the resulting ssDNA. This results in the formation of a single HJ which is resolved by the cleavage of the crossed strand. Although this pathway may not have immediate relevance for DSB-induced genome engineering, it is biologically significant for repairing breaks that lack a second end necessary for DSBR or SDSA.
8. Conclusion
In conclusion, the field of genetics, CRISPR, and gene editing has undergone a transformative change in recent years. The rapid advancements in technology and knowledge in this field have paved the way for remarkable achievements in research and applications. With this new knowledge and techniques, we have the potential to revolutionize the field of medicine and healthcare, allowing us to treat and even cure previously incurable genetic diseases. However, the ethical implications of gene editing cannot be ignored, and there must be a careful consideration of the consequences of this technology. As the technology continues to improve and become more accessible, it is likely that we will see wider and more significant use of gene editing in the coming years. The potential benefits are vast, but it is important to proceed with caution and ensure that the use of this technology is regulated in an ethical and responsible manner.
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- https://www.nature.com/articles/s41576-021-00386-0
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981265/
- https://bdsc.indiana.edu/stocks/misc/hack.html
- https://nigms.nih.gov/education/factsheets/Pages/pharmacogenomics.aspx
- https://www.biorxiv.org/content/10.1101/2022.03.25.485874v1.full
- https://www.pnas.org/doi/10.1073/pnas.1321030111
