Introduction
The landscape of healthcare is rapidly evolving, with molecular medicine poised to revolutionize traditional practices. Groundbreaking advancements in our understanding of genetics and ongoing research have paved the way for methodologies capable of directly modifying DNA sequences. This capability, known as genome editing, has emerged from earlier, less precise techniques like nuclease technologies and chemical methods. Initial genome editing technologies, such as meganucleases, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), represented significant progress. However, these early tools were hampered by limitations, notably lower specificity and undesirable off-target effects. Furthermore, efficient delivery methods into host cells presented a considerable hurdle from a biotechnology standpoint. The advent of small RNAs, including microRNA (miRNA) and small interfering RNA (siRNA), offered alternative research tools, reducing reliance on animal models and cell lines. The more recent discovery of CRISPR/Cas9 technology has marked a paradigm shift, promising enhanced efficiency, greater feasibility, and a broader spectrum of clinical applications. This revolutionary technology is propelling genome engineering into a new era of molecular precision. This review aims to delve into the intricacies of various genome editing tools, providing a detailed comparison and contrast of their mechanisms of action, advantages, and limitations, ultimately focusing on their potential and challenges in the rapidly advancing field of molecular medicine.
Over the past fifty years, since the groundbreaking discovery of the DNA double helix, molecular biology has witnessed an unprecedented surge in technological innovation. These advancements are now converging towards practical applications in both clinical and laboratory settings.1 The convergence of high-throughput sequencing technologies, deepened insights into the intricate workings of genetic mechanisms, and the development of user-friendly nanotechnologies have empowered scientists to manipulate genetic code with increasing precision at multiple levels.2 The last two decades have been particularly transformative, marked by the proliferation of molecular techniques that enable the editing of genes and the modulation of their pathways. For the first time, humanity possesses the tools to precisely edit DNA sequences and to influence mRNA fate through post-transcriptional modifications.3
Genome editing encompasses a diverse array of techniques aimed at modifying DNA sequences. These modifications can range from deletions and alterations in mRNA processing to post-transcriptional changes, all ultimately leading to altered gene expression and consequently, changes in protein function and cellular behavior.4, 5 Despite their diversity, these methods share fundamental steps: the delivery of the genetic tool into the cell and subsequently into the nucleus; the targeted alteration of gene transcription and processing; and the final outcome – a protein product that is suppressed, overexpressed, or functionally modified.6, 7 From a broader perspective, these techniques, while conceptually simple, involve complex processes including receptor-ligand interactions, diverse cell entry mechanisms such as lipofection, sonication, and transfection, and a cascade of downstream pathway effects. The efficacy and applicability of these technologies are further nuanced by factors such as specificity, sensitivity, off-target effects, cost-effectiveness, and the level of technical expertise required. Moreover, the body’s immune response to foreign genetic material introduces another layer of complexity, potentially leading to rejection, particularly in therapeutic contexts.
To fully realize the potential of genome editing, a comprehensive understanding is crucial. This includes not only the methodological differences between various techniques but also the identification of targetable diseases, the development of innovative nanotechnology tools to enhance gene editing, and careful consideration of the ethical implications. The platforms supporting these technologies are in constant evolution, driven by technological miniaturization, automation, and ongoing research aimed at improving the efficiency and specificity of gene editing outcomes. Alongside these technological advancements, the establishment of robust regulatory frameworks and standardized protocols is essential. This will be critical for minimizing variability between and within methods, clearly defining the appropriate applications and limitations of each technique, and ultimately facilitating the advancement of personalized medicine.
This review provides a concise overview of prominent genome editing technologies, offering a comparative analysis of their strengths and weaknesses. It further explores recent advancements in the field and addresses some of the pressing bioethical concerns associated with these powerful tools.
Review Methodology
To ensure a comprehensive and up-to-date review, we conducted extensive searches on PubMed, focusing on articles published within the last 10 and 5 years using the keywords “genome-editing techniques” or “gene-editing techniques.” These searches yielded 4,466 and 4,054 references, respectively. To narrow our focus to specific genome editing methods, we performed targeted searches for “conventional genome-editing systems” (n = 100), “chemical methods” (n = 252), “meganucleases” (n = 83), “zinc-finger nucleases (ZFNs)” (n = 890), “transcription activator-like effector nucleases (TALENs)” (n = 1,136), “homing endonucleases” (n = 265), and “CRISPR” (n = 11,421). Given the vast literature on CRISPR, our search was refined to reviews providing conceptual information on common techniques and comparative analyses of ZFNs, TALENs, and CRISPR technologies. Finally, we explored the literature for emerging gene editing methods and the associated bioethical considerations arising from genome biotechnologies. This multi-faceted search strategy allowed us to synthesize a review that is both broad in scope and detailed in its comparative analysis of key genome editing tools.
Genome-Editing Techniques: An Overview
The rapid progress in biotechnology has provided profound insights into the biochemical and molecular mechanisms governing DNA editing and subsequent pathway modifications. Numerous biotechnologies have demonstrated potential for clinical translation, and the field of genome editing is characterized by continuous evolution and refinement. While newer techniques are showing remarkable promise, established methods are also undergoing continuous updates and improvements. For clarity and conciseness, Figure 1 provides a consolidated overview of the major genome editing techniques.
Figure 1.
A Consolidated Overview of Genome-Editing Techniques
This figure visually summarizes the diverse landscape of genome editing tools, highlighting the evolution from early methods to the more recent and advanced CRISPR-Cas systems. The techniques depicted range from chemical methods to nuclease-based approaches, including Meganucleases, ZFNs, TALENs, and CRISPR-Cas, providing a comprehensive snapshot of the field.
We will now discuss representative genome editing techniques in more detail, focusing on their mechanisms and applications.
Meganucleases: Precision Enzymes for Genome Editing
Meganucleases are naturally occurring enzymes known for their exceptional specificity in recognizing and cleaving DNA sequences. These enzymes target long DNA sequences (12-40 base pairs), which are statistically rare in the genome, thus minimizing off-target effects. Their high specificity arises from their unique protein structure that precisely interacts with the DNA target. However, the very specificity of meganucleases also presents a challenge. Designing a meganuclease for a novel target site is a complex protein engineering task, limiting their widespread applicability and making them less adaptable for routine genome editing tasks compared to more programmable tools.
Zinc-Finger Nucleases (ZFNs): Customizable DNA Binding Domains
Zinc-finger nucleases (ZFNs) represent a significant step forward in genome editing technology. ZFNs are artificial restriction enzymes engineered by fusing a zinc-finger DNA-binding domain to a DNA-cleavage domain, typically from the FokI endonuclease. Zinc fingers are protein motifs that can be designed to recognize specific DNA sequences. By linking arrays of zinc fingers, researchers can create ZFNs that target unique genomic loci. The FokI nuclease domain requires dimerization to become active, meaning two ZFNs must bind to opposite strands of the DNA near the target site for cleavage to occur. This dimerization requirement enhances specificity and reduces off-target cleavage.
Figure 2.
Excision of Selective Site of dsDNA by Utilizing Artificial Restriction DNA Cutter
Figure 2 illustrates the mechanism of ZFN-mediated DNA cleavage. Two ZFN molecules are engineered to bind to specific DNA sequences flanking the desired editing site. The FokI nuclease domains then dimerize, creating a double-strand break at the targeted location. This break initiates the cell’s DNA repair mechanisms, which can be harnessed for gene knockout or gene insertion.
Transcription Activator-Like Effector Nucleases (TALENs): Highly Programmable DNA Binding
Transcription activator-like effector nucleases (TALENs) are another class of engineered nucleases that offer improved programmability compared to ZFNs. TAL effectors are proteins secreted by Xanthomonas bacteria that bind to DNA in a sequence-specific manner. The DNA-binding domain of TAL effectors consists of a series of repeated modules, each recognizing a single DNA base pair. This modularity allows for the straightforward design of TALENs to target almost any DNA sequence. Similar to ZFNs, TALENs are constructed by fusing a TAL effector DNA-binding domain to the FokI nuclease. The requirement for dimerization of FokI domains again contributes to high specificity.
Figure 5.
Diagram Showing Mechanisms of Transcription Activator-like Effector Nucleases
Figure 5 provides a schematic representation of TALEN-mediated genome editing. TALEN proteins are engineered to recognize specific DNA sequences. Upon binding to the target site, the FokI nuclease domains dimerize and induce a double-strand break. This break is then repaired by cellular mechanisms, enabling precise genome modifications.
CRISPR/Cas9: Revolutionizing Genome Editing with RNA Guidance
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system represents a revolutionary advancement in genome editing. Unlike ZFNs and TALENs that rely on protein-DNA interactions for target recognition, CRISPR/Cas9 utilizes RNA-DNA complementarity. The system consists of two key components: the Cas9 nuclease, which is responsible for DNA cleavage, and a guide RNA (gRNA), a short RNA sequence that directs Cas9 to the specific DNA target. The gRNA is designed to be complementary to the target DNA sequence, allowing for highly flexible and easily programmable targeting. This programmability and simplicity have made CRISPR/Cas9 widely adopted in research and therapeutic applications.
Figure 6.
Schematic Demonstrating the Concept of CRISPR/Cas9 Interactions Leading to the Destruction of Viral Genome at the Selected Splice Site by the crRNA/gRNA
Figure 6 illustrates the mechanism of CRISPR/Cas9 system. The guide RNA (gRNA) directs the Cas9 nuclease to a specific DNA sequence. Cas9 then creates a double-strand break at the targeted site. This system has been adapted for a wide range of genome editing applications, from gene knockout to precise gene insertion and base editing.
Comparative Analysis of Genome Editing Tools: ZFNs, TALENs, and CRISPR/Cas9
While meganucleases, ZFNs, TALENs, and CRISPR/Cas9 all offer powerful capabilities for genome editing, they differ significantly in their design complexity, specificity, efficiency, and applicability. Tables 1, 2, and 3 provide a detailed comparative assessment of ZFNs, TALENs, and CRISPR/Cas9, highlighting their biotechnological differences, side effect profiles, and clinical and research applications.
Table 1.
Biotechnology Differences among Prototype Genome-Editing Techniques
| Serial No. | Parameter | ZFN | TALEN | CRISPER/Cas | Reference |
|---|---|---|---|---|---|
| 1 | Design Simplicity | Moderate (ZFNs need customized protein for every DNA sequence) | Slightly Complex (identical repeats are multiple, which creates technical issues of engineering and delivery into cells) | Simpler (available versions for crRNA can be easily designed) | 48 |
| 2 | Engineering Feasibility | Low | Higher | Highest | 24, 49 |
| 3 | Multiplex Genome Editing | Few models | Few models | High-yield multiplexing available (no need for obtaining embryonic stem cells) | 48, 50 |
| 4 | Large-Scale Library Preparation | Not much progress (need individual gene tailoring) | Not much progress (need individual gene tailoring) | Progress demonstrated (CRISPR only requires plasmid containing small oligonucleotides) | 51 |
| 5 | Specificity | Low | Higher | Highest | 24 |
| 6 | Efficiency | Normala | Normalb | High | 24, 48, 52 |
| 7 | Cost | Low | High | Low | 53 |
aSome new versions are more efficient24, 48 but CRISPR science is evolving more.
bCpf1 protein addition will probably improve cell delivery methods.51, 52
Table 1 summarizes the key biotechnological differences. CRISPR/Cas9 stands out for its superior design simplicity and engineering feasibility. The RNA-guided targeting of CRISPR/Cas9 is significantly easier to implement compared to the protein engineering required for ZFNs and TALENs. Furthermore, CRISPR/Cas9’s multiplexing capability and suitability for large-scale library preparation make it a more versatile tool for high-throughput research and applications.
Table 2.
Side Effect Profiles for Genome-Editing Methods
| Serial No. | Parameter | ZFN | TALEN | CRISPER/Cas | Reference |
|---|---|---|---|---|---|
| 1 | Off-target effect incidence | – | – | – | 54 |
| a | Homologous recombination rate frequency | + | + | + | – |
| b | Non-homologous end joining (NHEJ) mutation rates | + | + | ++ (only with earlier versions) | 55, 56 |
| c | Immune reaction susceptibility | Less | Less | More | 57, 58 |
| d | RGEN-induced off-target mutagenesis | − | − | ++ | 59 |
| 2 | Cytotoxicity chances | ++ | + | + | – |
Table 2 compares the side effect profiles. While all three technologies can induce off-target effects, earlier versions of CRISPR/Cas9 were associated with higher rates of non-homologous end joining (NHEJ) mutations and RNA-guided endonuclease (RGEN)-induced off-target mutagenesis. However, advancements in CRISPR/Cas9 technology, such as the development of high-fidelity Cas9 variants and optimized guide RNA design, have significantly reduced off-target effects. Interestingly, CRISPR/Cas9 may elicit a stronger immune response compared to ZFNs and TALENs, which is an important consideration for in vivo therapeutic applications.
Table 3.
Clinical and Research Applications across Important Genome-Editing Techniques
| Serial No. | Parameter | ZFN | TALEN | CRISPER/Cas | Reference |
|---|---|---|---|---|---|
| 1 | Diagnostic Utility | + | + | +++ | 60 |
| 2 | Clinical Trial Use | ++ | + | +++ | 61 |
| 3 | Utility as Epigenetic Marker | ++ | +++ | ++++ | 62 |
| 4 | Making Gene-Knockout Models for Research | No | No | Yes (CRISPRi) | 63 |
| 5 | Capacity for Modification of Mitochondrial DNA | No | No | Probable | 64 |
| 6 | Genetic Editing in Human Babies | No | No | Yes | 65 |
| 7 | RNA Editing | No | No | Yes | 66 |
Table 3 highlights the clinical and research applications. CRISPR/Cas9 exhibits superior utility across various applications, including diagnostics, clinical trials, epigenetic marking, gene knockout model generation (CRISPRi), mitochondrial DNA modification, and even RNA editing. While ZFNs and TALENs have shown promise in certain clinical applications, CRISPR/Cas9’s versatility and efficiency have rapidly propelled it to the forefront of both research and therapeutic genome editing.
Advancements in Genome Engineering: Beyond Traditional Nucleases
The field of genome engineering is constantly evolving, with exciting new modalities emerging that go beyond traditional nuclease-based approaches. Oligodeoxyribonucleotides (ODNs) are being explored in combination with double-stranded transcription factor decoys (TFDs) as therapeutic agents for various diseases. This approach targets transcription factors, modulating gene expression and downstream protein activity.37 Furthermore, research by Papaioannou et al.38 demonstrated the use of single-stranded ODNs for precise, footprint-free genome editing of point mutations. This innovative technique employs a drug-inducible Cas9 transgene delivered via a transposon, enabling highly specific and efficient Cas9-mediated genome editing without the need for conventional donor templates, thus minimizing off-target effects and enhancing safety.
Other novel genome editing strategies are also under development. Martínez-Gálvez et al.39 have shown that using single-stranded DNA (ssDNA) and argonautes can improve gene editing efficiency. Looking ahead, enzymes like integrases might further refine genome editing by potentially eliminating the need for nucleases altogether.40
Perhaps the most transformative concept in genome engineering is whole genome synthesis, which envisions the de novo creation of genomes based on designed DNA sequences. This ambitious concept, termed synthetic genomics, could revolutionize our approach to genetic manipulation.41 While still in its early stages, synthetic genomics holds the potential to eventually surpass traditional genome editing techniques, offering unprecedented control over the genetic makeup of organisms.
Bioethical Issues and Genome-Editing Techniques: Navigating the Ethical Landscape
The immense power of genome editing tools carries significant bioethical implications. While offering tremendous potential for advancements in crop development and human health, these technologies also raise concerns about misuse, particularly in germline genetic manipulation. Experts have voiced genuine ethical concerns that need careful consideration.42 The long-term societal impact of genome editing is yet to be fully understood, and potential unintended consequences for future generations must be addressed proactively.43 Key ethical considerations include the morality of germline editing, the potential for eugenics and exacerbating social inequalities, informed consent in clinical applications, religious perspectives, and the specter of “designer babies” and genetic enhancement leading to “superhumans”.44, 45, 46 Moreover, the potential for genome editing to be weaponized raises serious security concerns.47
While the desire for healthy offspring and access to the best available treatments are widely recognized, the unfolding genome editing revolution necessitates careful and regulated translation of these technologies into molecular medicine and other sectors, such as agriculture and food production. This requires a broad societal dialogue involving public engagement, expert discussions, the participation of biotechnologists and bioethicists, the establishment of regulatory frameworks within legislative bodies, and the development of clear guidelines and oversight mechanisms for permissible applications. A balanced and ethically informed approach is crucial to harness the benefits of genome editing while mitigating potential risks.
Conclusions
This review has explored the diverse landscape of genome editing technologies, encompassing their classification, mechanisms of action, comparative analysis, recent advancements, and critical bioethical considerations. CRISPR/Cas technologies have emerged as a dominant force, surpassing ZFNs and TALENs in many aspects. However, CRISPR/Cas systems are also undergoing continuous refinement, with ongoing innovations aimed at enhancing their capabilities and minimizing off-target effects. The relentless pace of innovation in gene modification technologies suggests that even CRISPR/Cas may eventually be superseded by even more advanced approaches, potentially including synthetic genomics. Amidst these groundbreaking developments, it is imperative to prioritize and address the significant bioethical challenges posed by genome editing to ensure responsible and beneficial applications of these powerful tools.
References
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[6] …
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[37] …
[38] …
[39] …
[40] …
[41] …
[42] …
[43] …
[44] …
[45] …
[46] …
[47] …
[48] …
[49] …
[50] …
[51] …
[52] …
[53] …
[54] …
[55] …
[56] …
[57] …
[58] …
[59] …
[60] …
[61] …
[62] …
[63] …
[64] …
[65] …
[66] …
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