With the continuous development of biotechnology and genetic engineering, gene editing technology has become one of the most cutting-edge and hottest research areas. Gene editing technology is a precise modification technique targeted at DNA sequences (Ren et al., 2019), possessing advantages such as high efficiency, accuracy, and strong controllability. It is widely applied in biomedical research, agriculture, animal husbandry, and other fields.

In the field of human disease treatment, gene editing technology can achieve the precise modification of patient gene sequences, enabling the treatment or cure of diseases and contributing to the advancement of human health. In agriculture and animal husbandry, gene editing technology allows for the regulation of plant and animal genes, enhancing crop and animal productivity, quality, and disease resistance, thereby contributing to agricultural production and food security. In biological science research, gene editing technology provides a powerful tool for scientists to explore gene functions, discover new drug targets, and unravel the mysteries of life.

The classification and principles of gene editing technology are important components of gene editing research. Gene editing technology consists of nuclease-based techniques, CRISPR-Cas system-based techniques, transcription activator-based techniques, and chemical modification-based techniques. Although these techniques use different tools and methods for modifying DNA sequences, their principles are centered around targeted cleavage of DNA sequences, leading to the creation of double-strand breaks. Subsequently, these breaks are repaired using the innate DNA repair mechanisms, thereby accomplishing modifications in the target genes.

This review aims to provide an overview of the classification and principles of gene editing technology. It introduces the principles and applications of different types of gene editing technologies, analyzes their advantages and challenges, and explores the future prospects of gene editing technology. It is hoped that this review will serve as a reference and guide for the research and application of gene editing technology, making contributions to the advancement of gene editing technology.

1 Gene Editing Technology Classification

1.1 Basic classification of gene editing technology

Gene editing technology is a precise editing technique for DNA sequences with broad application prospects. Gene editing technology can be classified into basic and advanced categories. The basic classification includes nuclease-based techniques, chemical modification-based techniques, and RNA interference-based techniques.

Nuclease-based techniques primarily encompass zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). The ZFN technology utilizes artificially synthesized zinc finger proteins and FokI nucleases to achieve targeted cleavage of DNA sequences. TALEN technology, on the other hand, employs artificially synthesized transcription activator-like effectors and FokI nucleases for targeted cleavage of DNA sequences. Both of these techniques demonstrate high levels of specificity and accuracy, but they involve higher manufacturing costs and necessitate professional technical support.

Chemical modification-based techniques mainly include locked nucleic acids (LNAs) and antisense nucleotides. LNA is a technology that utilizes chemical modifications of DNA sequences to inhibit the expression of specific genes. On the other hand, antisense nucleotides involve the synthesis and introduction of RNA molecules that are complementary to the target RNA, thereby achieving gene expression inhibition. These techniques have the advantages such as low manufacturing costs and simplicity of operation. However, they have the disadvantage of non-specificity, which may lead to impacts on other genes.

RNA interference-based techniques mainly include siRNA and shRNA. siRNA is a short RNA molecule that can interact with target RNA and induce its degradation. On the other hand, shRNA is a long chain RNA molecule that can guide target RNA to bind to RNA-dependent RNA polymerase, leading to its degradation. These techniques exhibit high specificity and accuracy. However, it is necessary to identify suitable RNA sequences to achieve gene expression inhibition.

In summary, nuclease-based, chemical modification-based, and RNA interference-based techniques are the basic classifications of gene editing technology. Each of these techniques has its own advantages and disadvantages, and selecting the appropriate technique for different application scenarios can achieve better gene editing and regulation.

1.2 Advanced classification of gene editing technology

The advanced classification of gene editing technology includes techniques based on the CRISPR-Cas system (Yang et al., 2019) and techniques based on transcription activator factors.

Transcription activator factor-based technologies primarily include CRISPR-dCas9 and TALE-TF. These techniques utilize the DNA-binding capability of transcription activator factors to achieve gene regulation. The CRISPR-dCas9 technology uses the DNA-binding capability of the Cas9 protein without cleaving the DNA sequence, but instead binds with transcription activator factors to activate genes. TALE-TF technology, on the other hand, uses the combination of transcription activator factors and artificially synthesized TALE proteins to achieve gene regulation.

Techniques based on the CRISPR-Cas system are currently one of the most popular gene editing technologies. CRISPR-Cas9 technology utilizes the pairing recognition mechanism between the Cas9 protein and RNA molecules to precisely cleave DNA sequences. This technology demonstrates high specificity and accuracy, and it is relatively cost-effective to manufacture. Therefore, it has found widespread applications in biomedical research, agriculture, animal husbandry, and other fields. Furthermore, the techniques based on the CRISPR-Cas system have led to the development of a series of technologies, such as dCas9-FokI and CRISPRa/Cas9, which enable targeted DNA sequence modifications and gene regulation.

In summary, techniques based on transcription activator factors and techniques based on the CRISPR-Cas system are advanced classifications of gene editing technology. These techniques exhibit characteristics such as high efficiency, accuracy, and strong control ability, providing new insights and methods for human disease treatment and biological research. However, these technologies also face challenges related to improving technical safety and reliability, as well as ethical concerns.

1.3 Applications of gene editing in medical research

Gene editing technology can be used in various aspects of medical research, including gene function studies, disease modeling, and gene therapy. Here are some applications of gene editing in medical research. Firstly, disease modeling: Gene editing techniques can be utilized to create disease models to investigate disease mechanisms and treatment strategies. For instance, scientists have employed CRISPR/Cas9 technology to generate models of human genetic diseases such as cystic fibrosis and amyotrophic lateral sclerosis in mice and monkeys. Secondly, gene function studies: Gene editing technology can be employed to study gene functions and regulatory mechanisms. Scientists can study the roles of specific genes in cell growth, differentiation, and function by deleting or modifying them. Gene editing technology can also be applied in gene therapy, where it is used to treat certain genetic diseases, such as cystic fibrosis and hereditary blindness (Niu et al., 2019). Scientists can repair or replace defective genes using gene editing techniques, restoring normal cellular functions. This approach is known as gene therapy. Gene editing technology can be used in the treatment of certain types of tumors as well. For example, scientists can enhance the immune cells' ability to attack tumors using gene editing techniques, leading to the therapeutic effect in tumor treatment.

In summary, gene editing technology has extensive applications in medical research, enabling scientists to better understand the mechanisms of disease occurrence and development. It also provides new insights and methods for disease treatment and prevention. With the continuous development and improvement of the technology, the application and research of gene editing will continue to deepen and expand.

2 Principle of Gene Editing Technology

2.1 Principle of ZFN technology

The zinc finger nuclease (ZFN) technology is a gene editing technique that utilizes the DNA-binding ability and nuclease cleavage activity of nucleases to achieve precise gene editing (Lu et al., 2018). The ZFN technology primarily includes two components: artificially synthesized zinc finger proteins and the FokI nuclease.

Zinc finger proteins are proteins that can bind to DNA and exhibit high specificity. Through artificial design, they can be precisely bound to the target DNA sequence. Each zinc finger protein typically contains three zinc finger domains, with each domain responsible for recognizing and binding to three base pairs in the DNA. By synthesizing and assembling different zinc finger domains, specific recognition and binding to any DNA sequence can be achieved.

FokI nuclease is a type of double-stranded cleavage enzyme that can cut DNA strands, thereby enabling precise editing of DNA sequences. In ZFN technology, two zinc finger proteins form a dimer with FokI nuclease. One zinc finger protein recognizes and binds to the target DNA sequence, while the other zinc finger protein recognizes and binds to another targeted DNA sequence. FokI nuclease mediates the cleavage and recombination of the two DNA strands, thus achieving precise editing of DNA sequences.

ZFN technology offers high specificity and accuracy, enabling modification and editing of any DNA sequence within the genome. Additionally, ZFN technology can be combined with other gene editing techniques, such as TALEN technology and the CRISPR-Cas system, to further enhance its editing efficiency and specificity. However, ZFN technology also faces certain challenges and issues, including high preparation costs, technical complexity, and the need to improve editing efficiency and specificity. Therefore, when employing ZFN technology for gene editing, careful consideration of its advantages and disadvantages, limitations, and applicability is necessary. Combined application with other gene editing techniques should be considered to achieve precise editing and regulation of genes.

2.2 Principle of TALEN technology

TALEN technology is a technique that utilizes Transcription Activator-Like Effector Nucleases (TALENs) to achieve gene editing (Shen et al., 2013). TALEN technology mainly includes two parts of TALEN proteins and the FokI nuclease (Figure 1).

Figure 1 TALEN technology

TALEN proteins are fusion proteins composed of an N-terminal transcription activator-like effector (TALE) domain and a C-terminal FokI nuclease domain. The TAL domain exhibits high specificity and can recognize and bind to individual nucleotides in DNA, enabling recognition and binding to any DNA sequence. By synthesizing and assembling different TAL domains, specific recognition and binding to any DNA sequence can be achieved, thus achieving precise editing of specific sequences within the genome.

FokI nuclease is a type of double-stranded cleavage enzyme that can cut DNA strands, enabling precise editing of DNA sequences. In TALEN technology, two TALEN proteins combine to form a dimer. One TALEN protein can recognize and bind to the target DNA sequence, while the other TALEN protein can recognize and bind to another targeted DNA sequence. The FokI nuclease then mediates the cleavage and recombination of the two DNA strands, thus achieving precise editing of DNA sequences.

TALEN technology exhibits high specificity and accuracy, allowing for modification and editing of any DNA sequence within the genome. Additionally, TALEN technology can be combined with other gene editing techniques such as ZFN technology and the CRISPR-Cas system, further enhancing its editing efficiency and specificity.

However, TALEN technology also faces similar issues as ZFN technology. Therefore, when using TALEN technology for gene editing, careful consideration of its advantages, disadvantages,  limitations, and applicability is equally necessary. Comprehensive application in combination with other gene editing techniques should be considered as well.

2.3 Principle of CRISPR/Cas9 technology

CRISPR/Cas9 is a gene editing technology that utilizes CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequences and Cas9 (CRISPR-associated Protein 9) protein. CRISPR/Cas9 technology primarily consists of two components: sgRNA (single guide RNA) and Cas9 protein.

sgRNA is an artificially synthesized RNA molecule composed of CRISPR sequences and an RNA sequence that is complementary to the target DNA sequence. CRISPR sequences are commonly found DNA sequences in bacteria and archaea, which can form RNA-DNA duplexes with foreign viral or plasmid DNA, thereby providing immune defense. In CRISPR/Cas9 technology, the sgRNA binds to the Cas9 protein to form a complex. The RNA sequence of sgRNA can recognize and bind to the target DNA sequence, while the Cas9 protein mediates the cleavage and recombination of DNA strands, enabling precise editing of DNA sequences.

Cas9 protein (Figure 2) is a type of double-stranded cleavage enzyme that can cut DNA strands, enabling precise editing of DNA sequences. In CRISPR/Cas9 technology, the Cas9 protein recognizes and binds to the target DNA sequence by associating with the sgRNA, thereby mediating the cleavage and recombination of the DNA sequence. Cas9 protein possesses high specificity and accuracy, allowing for modification and editing of any DNA sequence within the genome.

Figure 2 Schematic representation of the major domains of Cas9 protein

CRISPR/Cas9 technology possesses high specificity, accuracy, and efficiency, enabling precise editing and regulation of any DNA sequence within the genome. Additionally, CRISPR/Cas9 technology can be combined with other gene editing techniques such as TALEN and ZFN to further enhance its editing efficiency and specificity. However, CRISPR/Cas9 technology also encounters certain issues and challenges, such as off-tumor recognition and inconsistent editing efficiency (Li et al., 2018). Therefore, caution is necessary when utilizing CRISPR/Cas9 technology for gene editing.

2.4 Principles of other gene editing techniques

Gene editing techniques is a technology that enable precise editing and regulation of the genome. In addition to ZFN, TALEN, and CRISPR/Cas9 technologies, there are several other gene editing techniques. These techniques differ from the aforementioned three technologies but can also achieve precise editing and regulation of the genome.

Gene knockout technology is a common gene editing technique (Guo et al., 2019). Gene knockout technology achieves the suppression and elimination of the target gene through methods such as RNA interference (RNAi) or transcription factor inhibitors. RNAi technology involves the introduction of artificially synthesized small RNA molecules that bind complementarily to the target mRNA, leading to mRNA degradation and suppression of gene expression. Transcription factor inhibitors work by inhibiting the activity of transcription factors, thereby reducing the expression level of the target gene. Although gene knockout technology cannot directly edit the gene sequence, it remains an important gene editing technique that provides valuable information for gene function research.

Another gene editing technique is gene replacement technology. Gene replacement technology involves the replacement of the target gene sequence with a synthetic DNA sequence, thereby achieving precise editing and regulation of the genome (Wang et al., 2017). Gene replacement technology can be applied in areas such as gene therapy and treatment of genetic diseases. Gene replacement technology can be achieved through different methods, including CRISPR/Cas9-mediated gene replacement technology and homologous recombination.

In addition, there are several other gene editing techniques, such as anti-sense RNA technology and gene repair technology. Anti-sense RNA technology involves the introduction of synthetically created anti-sense RNA molecules that bind complementarily to the target mRNA, leading to mRNA degradation and inhibition of gene expression. Gene repair technology, on the other hand, involves the introduction of synthetically created DNA sequences that undergo homologous recombination with the target DNA sequence, thereby achieving precise editing and repair of the genome. Overall, although these techniques have their own characteristics, they all enable precise editing and regulation of the genome, providing strong support for gene function research, gene therapy, and treatment of genetic diseases.

3 Application of Gene Editing Techniques in Human Disease Treatment

3.1 Application in cancer treatment

Cancer is a severe disease that causes suffering and death for many individuals worldwide. However, with the continuous advancement of scientific technology, gene editing techniques have gradually gained potential and applications in cancer treatment. Gene editing techniques, particularly the CRISPR-Cas9 system, can accurately modify gene sequences, providing new ideas and tools for cancer treatment. For instance, specific mutations in certain types of cancer can be repaired using the CRISPR-Cas9 system to restore normal cellular proliferation and differentiation signaling mechanisms. Furthermore, gene editing techniques can also be used to enhance or activate a patient's immune system to boost their resistance against cancer. By editing the patient's own immune cells, they can better identify and eliminate tumor cells. For example, the use of the CRISPR-Cas9 system can edit the genes of T-cells, equipping them with stronger anti-tumor abilities. Gene editing techniques can also be employed to alter the surface markers of tumor cells, making them more recognizable and vulnerable to attack by the immune system.

Gene editing techniques in cancer treatment are still in the early stages of research, and there are challenges and limitations that need to be addressed. For example, the precision and efficiency of gene editing techniques still need further improvement to avoid unintended effects on normal cells. Ethical and safety issues also need to be carefully considered to ensure the safety and feasibility of treatments. Overall, the application of gene editing techniques in cancer treatment offers potential hope and opportunities for patients. By precisely repairing or modulating cancer-related genes, gene editing techniques can provide new avenues for individualized treatment.

3.2 Application in the treatment of immunodeficiency disorders

The normal functioning of the immune system is vital for our overall health. However, some individuals may suffer from immunodeficiency disorders due to genetic mutations, where their immune system fails to function properly, making them susceptible to infections. In recent years, the development of gene editing techniques has provided new opportunities for the treatment of immunodeficiency disorders. Gene editing techniques have brought new breakthroughs for the treatment of immunodeficiency disorders. One common immunodeficiency disorder is primary immunodeficiency disease (PID), which includes Wiskott-Aldrich Syndrome (WAS), Severe Combined Immunodeficiency (SCID), among others. Using gene editing techniques, it is possible to correct the defects in disease-associated genes, restoring normal immune system function. One approach to applying gene editing techniques is by directly repairing mutations in the genes of immunodeficiency disorder patients. Through the guidance of the CRISPR-Cas9 system, researchers can precisely repair or restore abnormal portions of genes in patients. For example, in the case of WAS patients, who carry mutations in the WAS gene leading to immune cell dysfunction, gene editing techniques can be utilized to correct these mutations, allowing immune cells to function normally in immune responses. Furthermore, gene editing techniques can also enhance the immune system of patients by modifying the activity and function of immune cells. By editing the genes of immune cells, they can acquire stronger killing and infection-fighting abilities. For instance, in SCID patients, the CRISPR-Cas9 system can be used to edit the exon skipping gene to restore normal T cell function. This approach allows the patient's immune cells to regain normal activity, thus improving their ability to resist infections.

Despite demonstrating significant potential in the treatment of immunodeficiency disorders, gene editing technologies still face several challenges and limitations. For instance, the precision and efficiency of gene editing techniques require further improvement to ensure accurate and effective gene modifications. Moreover, ethical and safety issues need to be carefully addressed to ensure the safety and feasibility of treatments. In summary, the application of gene editing techniques in the treatment of immunodeficiency disorders offers new hope for these patients. By precisely repairing or enhancing the genes of individuals with immunodeficiency disorders, gene editing technologies can restore or strengthen the functionality of their immune systems.

3.3 Application in the treatment of other diseases

In the treatment of single-gene genetic disorders, gene editing technologies can achieve precise editing and regulation of target genes. For example, fragile X syndrome is a common single-gene genetic disorder (Wu et al., 2018). The CRISPR/Cas9 technology can be used to edit the genes associated with fragile X syndrome, thus providing a therapeutic approach for this disorder. Additionally, gene knockout techniques can also be applied to the treatment of single-gene genetic disorders.

Although gene editing technology holds vast potential for the treatment of human diseases, it currently faces technical and ethical challenges, such as off-target effects and safety concerns. Therefore, when utilizing gene editing technology for human disease treatment, careful consideration must be given to the advantages and limitations of the technique, as well as safety issues and the scope of its application. This is necessary to ensure accurate and effective gene editing to achieve optimal therapeutic outcomes.

4 Summary and Outlook

Gene editing technologies primarily include ZFN, TALEN, CRISPR/Cas9, and other techniques. In addition, there are several other gene editing techniques, such as gene knockout, gene replacement, anti-sense RNA, and gene repair. Each of these techniques has its own characteristics but can achieve precise editing and regulation of the genome. The principles of gene editing technology mainly involve editing and regulation of the genome through methods like DNA cleavage, DNA repair, and RNA interference.

The advantages of gene editing technology lie in its ability to achieve precise editing and regulation of the genome, providing powerful tools and means for gene function research and disease treatment. Gene editing technology can be applied for the treatment and prevention of various diseases, such as monogenic genetic disorders, cancer, and immunodeficiency disorders. Additionally, gene editing technology holds broad application prospects in precision medicine, agriculture, and biotechnology.

However, gene editing technology also faces several challenges (Liu et al., 2019), such as off-tumor recognition and safety concerns. Off-tumor recognition refer to the possibility of gene editing technology editing and regulating non-target genes, resulting in unintended side effects. Safety concerns arise from the potential consequences of gene editing technology, such as gene mutations and apoptosis, which may have implications for human health.

In the future, gene editing technology will continue to advance, but it is also necessary to address corresponding challenges and issues. To overcome these problems, continuous improvement and optimization of the technical details and operational procedures of gene editing technology are required to enhance its precision and safety. Furthermore, it is essential to strengthen the ethical review and regulation of gene editing technology to ensure its safety and legality in applications. The development of gene editing technology will bring more opportunities and challenges in fields such as gene function research, disease treatment, and biotechnology.

Author’s contributions

WW is the main author of the review, responsible for collecting and analyzing relevant literature, as well as writing the initial draft of the paper. I have read and approved the final manuscript.

Acknowledgments

I would like to express my gratitude to Lingfei Jin for providing valuable feedback on the paper revisions. The figures used in this paper are sourced from the internet, and I respect and uphold the rights of each image owner. If there are any copyright infringements related to the images, please feel free to contact me.

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