Research Status of Clustered Regulary Interspaced Short Palindromic Repeats Technology in the Treatment of Human Papillomavirus (HPV) Infection Related Diseases

Abstract

Background: CRISPR/Cas9 technology has rapidly advanced as a pivotal tool in cancer research, particularly in the precision targeting required for both detecting and treating malignancies. Its high specificity and low off-target effects make it exceptionally effective in applications involving Human Papillomavirus (HPV) related diseases, most notably cervical cancer. This approach offers a refined methodology for the rapid detection of viral infections and provides a robust platform for the safe and effective treatment of diseases associated with viral infections through gene therapy.

Purpose: Gene therapy, within this context, involves the strategic delivery of genetic material into target cells via a vector. This is followed by the meticulous modulation of gene expression, whether through correction, addition, or suppression, specifically honed to target tumor cells while sparing healthy cells. This dual capacity to diagnose and treat at such a precise level underscores the transformative potential of CRISPR/Cas9 in contemporary medical science, particularly in oncology and virology.

Research Design: This article provides an overview of the advancements made in utilizing the CRISPR-Cas9 system as a research tool for HPV-related treatments while summarizing its application status in basic research, diagnosis, and treatment of HPV.

Data Collection: Furthermore, it discusses the future prospects for this technology within emerging areas of HPV research and precision medicine in clinical practice, while highlighting technical challenges and potential directions for future development.

Keywords

Clustered Regulary Interspaced Short Palindromic Repeats/Cas system Human Papillomavirus cervical cancer

Background

The RNA-guided CRISPR-Cas nuclease system was originally found in bacteria as part of the adaptive immune system. Utilizing a guide RNA designed to recognize the 3-base-pair protospacer adjacent motif (PAM) sequence in the target DNA, the CRISPR/Cas9 genome editing technique is known for its exceptional ability to modify genetic components. This has led to a wide range of practical applications, such as base editing, transcriptional regulation, and epigenetic modification. With its remarkable power, specificity, and efficiency, the CRISPR-Cas gene editing tool allows for accurate and rapid screening of the entire genome, making gene therapy for specific diseases more accessible. In the realm of cancer research, the CRISPR-Cas system is utilized for genome editing to study the mechanisms underlying tumor development, metastasis, and occurrence.¹ Its application in treating HPV and other diseases is extensive.

Gene Editing Systems

In the field of life sciences, gene editing is a crucial technology that enables a thorough exploration of gene function in biological processes and has emerged as a promising therapeutic approach for specific diseases. Gene editing systems, which precisely manipulate DNA sequences by cleaving them, are widely used in the treatment of genetic disorders.

CRISPR, short for Clustered Regularly Interspaced Short Palindromic Repeats, is a key component in gene editing. The CRISPR sequences, along with CRISPR-associated (Cas) proteins, form the CRISPR/Cas system.² This system acts as a defense mechanism utilized by prokaryotes to protect themselves against foreign genetic material.³

Currently, the CRISPR/Cas system is the most commonly used gene editing tool, with Cas9 as its central component, followed by Cas12 and Cas13. CRISPR-Cas systems are categorized into 2 main groups, as outlined succinctly in Table 1.

Table 1.

Classification of CRISPR/Cas Systems⁴.

Class	Type	Effector Protein	Target
Class 1 CRISPR-cas systems	Type I	Multi-subunit complex (signature protein Cas3)	dsDNA, May exhibit collateral activity
	Type Ⅲ	Multi-subunit complex (signature protein Cas10)	DNA/RNA
	Type Ⅳ	Multi-subunit complex (signature protein Csf1)	dsDNA
Class 2 CRISPR-cas systems	Type Ⅱ	Cas9	Double-stranded DNA
	Type Ⅴ	Cas12	Double-stranded DNA, single-stranded DNA, May exhibit collateral activity
	Type Ⅴ	Cas14	Single-stranded DNA, May exhibit collateral activity
	Type Ⅵ	Cas13	Single-stranded RNA, May exhibit collateral activity

CRISPR/Cas Class 1 Systems

CRISPR-Cas class I systems encompass 3 distinct types: type I, type III, and type IV. These systems are distinguished by the presence of multiple effector proteins, which collectively utilize an effector module known as Cascade. This complex is responsible for recognizing and binding to prime-spacer adjacence-motif (PAM) sequences, and it unwinds the target DNA, facilitating interactions between the crRNA and its complementary DNA strands.^5,6 The Type III CRISPR-Cas system operates through multisubunit complexes, distinguished by the Cas10 protein, which has been demonstrated to participate in the activation of non-specific RNases Csm6 and Csx1.⁷ CRISPR-Cas systems, characterized by their presence in plasmids, continue to exhibit functions that are yet to be fully elucidated.^8,9

CRISPR/Cas Class 2 Systems

Class 2 CRISPR-Cas systems, notably type II alongside the rarer types V and VI, are distinguished by their streamlined architecture featuring a singular, large effector protein with multiple domains and functions.¹⁰ Their efficacy and simplicity have propelled these systems to the forefront of genetic engineering applications.¹¹

Employing the CRISPR-CasII system, the precision of genetic editing hinges on the guidance of Cas9 protein to DNA targets by a composite of crRNA (tracrRNA). This complex ensures the recruitment of Cas9 to the specific locus for gene alteration.¹¹ Diverging from this arrangement, the CRISPR-Cas type V system, encompassing variants such as V-A and V-B, is characterized by the Cas12 proteins (Cas12a, Cas12b, etc.). A notable distinction of this system lies in its streamlined requirement for only the Cas protein coupled with crRNA to facilitate genomic modifications.^12,13 In the context of genetic engineering, Cas12 is preferred over Cas9 owing to its compact structure, which enhances genomic fidelity by minimizing tolerances for mismatches between the target DNA sequence and the crRNA.¹⁴ Advancing further, the CRISPR-Cas type VI systems, which include subtypes VI-A to VI-D, feature Cas13 as their signature protein. Cas13’s distinct ability to discern single RNA strands underscores the prowess of the type VI systems.¹⁵ Unlike other systems, Cas13 utilizes a singular crRNA to hone in on RNA targets, creating precise blunt end cuts and indiscriminately degrading proximal single-stranded RNA.¹⁶

CRISPR/Cas9

The CRISPR/Cas9 system is derived from a bacterial defense mechanism against phage infection. This gene editing technology uses sgRNA for recognition and the Cas9 protein for precise genome targeting. It enables gene insertion or knockout through the host’s repair machinery.¹⁷ The CRISPR-Cas9 system can be classified into Type I, Type II, and Type III. Among these, the Type II system is characterized by a simpler composition, containing only the Cas9 protein. This protein includes the RuvC and HNH domains, which play a crucial role in precisely cleaving the target double-stranded DNA. The CRISPR-Cas9 technology is considered a groundbreaking system with transformative potential.

The CRISPR/Cas9 system exhibits exceptional precision and flexibility, rendering it a paramount choice in the realm of disease diagnosis and treatment. In comparison to conventional gene editing technologies, the CRISPR/Cas9 system boasts heightened accuracy and operability, enabling precise localization of target genomes, specific gene manipulation, and refined gene editing capabilities. Furthermore, this system possesses the capacity to activate or inhibit gene expression, thereby affording control over vital biological processes.

More precise and clinically safer variants in CRISPR-Cas type II systems involve genetically modified or evolved SpCas9 proteins, such as enhanced SpCas9 (eSpCas9), high-fidelity SpCas9-HF1, evoCas9, HypaCas9, and snip-Cas9,¹⁸ as well as certain homologous Cas9 proteins from other species (eg, Neisseria meningitidis Cas9,¹⁹ Staphylococcus aureus Cas9,²⁰ etc.). The latter possess more stringent PAM sequences, thereby reducing the potential for off-target sites during their functioning, and inherently have a lower ability to unwind DNA mismatched with the sgRNA.²¹

CRISPR/Cas12 and CRISPR/Cas13

As per a research paper released towards the end of 2019, CRISPR/Cas mechanisms have been categorized into 2 main classes, encompassing 6 distinct types and thirty-three subclasses.¹⁸ Class 1 comprises Type I, Type III, and Type IV systems, which possess effector modules consisting of intricate complexes with proteins such as Cas3, Cas5 to Cas8, Cas10, and Cas11 being part of this assembly. In contrast, Class 2 CRISPR/Cas systems encompass Type II, Type V, and Type VI, which are characterized by effector modules centered around a single, large protein with multiple domains, often accompanied by proteins like Cas9, Cas12, and Cas13.²²

The V-type CRISPR/Cas system is distinguished by its possession of a single RNA-directed RuvC nuclease domain and a solitary effector molecule.^23,24 Cas12a constitutes the initial characterized V-type CRISPR/Cas system, which preferentially targets and cleaves double-stranded DNA (dsDNA) and contiguous single-stranded DNA (ssDNA) sequences in a reversed orientation, requiring specific PAM sequences for activity. Cas12a, a bifunctional nuclease, exhibits both endonucleolytic and exonucleolytic enzymatic activities. This enzyme exhibits a preference for the generation of crRNA and the interference of target DNA, as well as the ability to cleave both cis- and trans-strands of single-stranded DNA. The single-stranded DNA (ssDNA)-binding Cas proteins exhibit a streamlined process for nucleic acid amplification and probe backcutting, necessitating fewer steps and culminating in intricately interwoven ends with a significantly reduced deletion frequency. As a secondary CRISPR/Cas system for mammalian genome editing, the Cas12a system has a high affinity for 5′-TTN PAM sequences, which allows a significantly expanded range of genomic targets to be recognized compared with the PAM sequences recognized by Cas9.²⁵

The CRISPR/Cas13 system, which belongs to the Class 1 Type VI CRISPR/Cas system, represents a recently discovered nuclease editing mechanism that exhibits remarkable specificity and efficiency in targeting RNA for site-specific editing, splicing regulation, downregulation, and elimination within cellular contexts. Unlike conventional CRISPR/Cas proteins, Cas13 functions as a multifaceted DNA enzyme that synergizes with RNA enzymes to achieve precise gene editing by selectively degrading RNA at the transcriptome level. Notably, Cas13 stands out as the smallest Type VI CRISPR-effector protein identified thus far, comprising merely 775 amino acids.

HPV

Introduction

Human Papilloma Virus (HPV), a distinctively epithelium-loving virus, holds the title as the most widely spread sexually transmitted infection globally.²⁵ Over 200 distinct strains of HPV have been identified, and 12 of them have been connected to cervical cancer cases. As per the IARC’s yearly report,²⁶ these viruses can be categorized into high-risk categories (types 16, 18, 31, 33, 35, 51, 52, 56, 58, and 59) and low-risk groups. Cervical cancer accounts for an estimated 91% of cancer-related deaths among women aged 15 to 44 years, making it the fourth most common cancer and the second most prevalent cancer in females.²¹ Furthermore, approximately 70% of all cervical cancer instances are directly connected to the highly oncogenic HPV types 16 and 18.

According to current research progress, there is currently no cure for HPV infection. However, preventive and therapeutic measures such as HPV vaccines, drug therapy, physical therapy, and surgery are available.²⁷

The early stages of HPV infection are typically asymptomatic, making early screening for HPV crucial in the prevention and treatment of cervical cancer. In recent years, various methods including fluorescence,²⁸ photochemistry,²⁹ microarray,³⁰ and electrochemical luminescence (ECL) have been developed for HPV detection.³¹ The majority of sensing strategies for HPV detection are often complex and time-consuming. Therefore, it is crucial to develop HPV detection methods that are simple to operate, accurate, highly sensitive, and cost-effective.

Traditional Treatment Methods

According to the relevant data, in addition to CRISPR technology, immunomodulatory therapy is the main means of non-surgical treatment of HPV. Immunomodulatory treatments are introduced below.

The primary objective of this approach is to modulate the immune system in order to target malignant tumors that are positive for HPV. The key strategy involves enhancing the immune system’s ability to recognize cancers associated with HPV by suppressing immune checkpoints and directly targeting viral antigens. Antigen-specific immune targeting is achieved through therapeutic vaccination and adoptive cell therapy.³² While epidemiological and genetic evidence strongly support the role of immunosuppression and immune resistance in the development of HPV-associated malignancies, immune checkpoint blockade (ICB) therapy has shown efficacy in treating these cancers in various settings and disease sites.

The current immunotherapy in clinical trials is the Listeria-derived HPV vaccine Lm-LLO-E7 (also known as AXAL or ADXS11 001), a live, bioengineered, single-nuclear, attenuated vaccine that produces an HPV16-E7 fusion protein that targets HPV-infected cells, and a vaccine that is engineered to secrete an HPV16-E7 fusion protein that targets HPV-infected cells. Sustained stimulation of innate and E7 antigen-specific adaptive immune responses. The phase Ⅰ/Ⅱ clinical trials of AXAL vaccine have demonstrated the anti-tumor activity and safety in the treatment of advanced and recurrent cervical cancer, and subsequent clinical trials are still in progress.^33,34

There is also a vaccine against viral oncoproteins E6 and E7 that is currently considered 1 of the most promising, ISA101, The vaccine consists of 9 overlapping long E6 peptides (5 32-mer E6 peptides and 4 25-mer E6 peptides) and 4 overlapping 35-mer E7 peptides (synthetic long-peptide HPV16 vaccine), which completely cover the HPV16-E6 and E7 oncoprotein sequences. Two independent studies have shown that more than 50% of patients with high-grade vulvar and vaginal intraepithelial neoplasia (VIN/VaIN) caused by HPV16 infection can produce strong HPV 16-specific immune responses to ISA101 vaccine.³⁵ This vaccine not only effectively blocks precancerous lesions, but also induces specific T cell immune responses targeting HPV in patients with advanced cervical cancer.^33,36

However, there are cases of cervical cancer caused by multiple HPV subtypes that are resistant to this treatment. In addition, the respective microenvironment of HPV-associated malignancies varies due to differences in the primary tumor location. In future research, it is essential to identify optimal strategies for enhancing antigen-specific immune responses to enhance oncology outcomes for patients.

Diagnosis and Treatment of HPV Based on CRISPR Technology

Related Therapeutic Targets

The oncoproteins E6 and E7 of HPV play a crucial role in promoting carcinogenesis and cancer cell proliferation, rendering them the primary therapeutic targets for cervical cancer. In preclinical and clinical trials, the CRISPR/Cas system has exhibited promising outcomes by specifically targeting and eliminating the oncogenes E6 and E7 from a patient’s genome, thereby offering potential control over the progression of HPV-associated cervical disease.^37-39

Detection Method Based on CRISPR Technology

Early detection by CRISPR technology is the typical Cas9 protein using the type II CRISPR-Cas system³⁷ or its modified nuclear fission null or dead Cas9 (dCas9) protein.³⁶ The breakthrough in CRISPR-based molecular diagnostics came with the discovery of the collateral protein activity of Cas12, Cas13, and Cas14, allowing for the amplification of specific targeting signals.¹³ Today, on the basis of CRISPR molecular detection method has a lot of improvement, also evolved a depends on the CRISPR/Cas V and VI protein with activity detection methods, but overall concept remains unchanged.¹⁰

In order to effectively prevent the occurrence of cervical cancer and improve postoperative follow-up treatment, rapid and reliable detection of HPV virus DNA is indispensable. The latest CRISPR-based detection of HPV primarily utilizes the Cas12 protein. The main methods include a CRISPR biosensing system based on SERS technology,⁴⁰ a CRISPR/Cas12a biosensing system based on upconversion luminescence resonance energy transfer,⁴¹ and a dual-channel system using CRISPR-Cas13a/Cas12a.⁴²

CRISPR biosensing system based on SERS technology

The Surface-enhanced Raman scattering (SERS) technology has been extensively utilized in the field of biological detection and analysis due to its exceptional sensitivity and the distinctive “fingerprint” characteristic exhibited by SERS spectra. By leveraging the alterations in SERS signals resulting from non-specific cleavage of single-stranded DNA (ssDNA) within the Cas12a/crRNA/DNA ternary complex system, virus detection can be achieved. Specifically, this method involves forming a ternary complex between the target viral nucleic acid, Cas12a, and crRNA to initiate cleavage of substrate single-stranded DNA ligation probe 1AuNPs@MBN@DNA1 as well as probe 2AuNPs@MBN@DNA2 within the cleavage system. The bridging effect between probes 1 and 2 induces changes in SERS signals, enabling determination of whether the tested sample contains the target nucleic acid. This sensing approach offers high sensitivity and rapid HPV gene detection capabilities that can be completed within a timeframe of 40 minutes.⁴⁰

CRISPR/Cas12a biosensing system based on upconversion luminescence resonance energy transfer

Utilizing a multi-stage high-temperature coprecipitation and shell epitaxial growth technique, the synthesis of NaYF4∶Yb3+, Er3+ (C-UCNPs) core layer and NaYF4@NaYF4∶Yb3+, Er3+@NaYF4 (CSS-UCNPs) inner shell upconversion nanomaterials with energy confinement were accomplished. The integration of these materials with the CRISPR/Cas12a-gold nanoparticle system enabled both colorimetric qualitative assessment and upconversion luminescence-based quantitative analysis of human papillomavirus DNA (HPV16 DNA), thus facilitating effective HPV detection. Importantly, this methodology showcases exceptional selectivity and can discern single base pair variations in HPV16 DNA, enhancing the tolerance for DNA fragment errors.⁴¹

Dual-channel system using CRISPR-Cas13a/Cas12a

The CRISPR-Cas13a/Cas12a double-channel system was developed for the rapid detection of HPV16/18. Firstly, the complete sequence of all HPV16/18 L1 genes was downloaded from NCBI, and sequence alignment was performed using the MAFFT software. Based on the relatively conserved region, HPV16/18-crRNA and HPV16/18-primers were designed. RAA primers were designed according to the conserved region of HPV16 L1 gene. The primer pairs were screened through RAA isothermal amplification experiments. Similarly, RAA primer pairs for the HPV18 L1 gene were also screened. Subsequently, a single-stranded DNA probe suitable for Cas12a protein in the dual-channel detection system was selected using CRISPR-Cas12a fluorescence detection method. A novel CRISPR-Cas13a/Cas12a-based dual-channel detection technology has been developed by integrating the CRISPR-Cas13a and CRISPR-Cas12a detection systems. This technology allows for swift detection of HPV16 through CRISPR-Cas13a nucleic acid detection and rapid detection of HPV18 utilizing CRISPR-Cas12a technology. Furthermore, a visual detection method based on CRISPR-Cas12a/Cas13a has been devised for simultaneous identification of both HPV16 and HPV18 viruses, enabling convenient on-site testing.⁴²

Treatment Based on CRISPR Technology

Among the various subtypes of HPV, HPV16 and HPV18 are dominant, so the related research mainly focuses on these 2 subtypes.

The HPV virus genome consists of 2 main segments: the early region E and the late region L. The early region E is essential for regulating viral replication and life cycle processes, while the late region L mainly encodes the capsid protein responsible for virus formation.⁴³ Within the HPV genome, the E6 and E7 genes are known as major oncogenes. The E6 oncogene acts by suppressing the p53 tumor suppressor pathway and hindering immune evasion by inhibiting RIG-I signal transduction,⁴⁴ while the E7 oncogene suppresses the activity of the retinoblastoma protein and impacts pathways involving p21 and other molecules.⁴⁵ Numerous studies have demonstrated that both E6 and E7 genes play pivotal roles in inducing apoptosis and cell cycle arrest.⁴⁶ Consequently, these genes represent key targets for investigating therapeutics related to HPV.

The work by Zhen et al implemented a CRISPR/Cas9 technology focusing on E6 and E7 genes,⁴⁵ evaluating its potential in HPV therapy by silencing these genes in SiHa cells. Our investigation has demonstrated the effective inhibition of cervical cancer progression in murine models by the CRISPR/Cas9 system. This was achieved through consistent suppression of HPV-16 E6 and E7 expression both in laboratory cultures and in vivo. Yoshiba et al performed experiments in which AAV sgE6 was injected into subcutaneous tumors and Cas9 gene was introduced into the high-risk HPV-positive cell lines HeLa, HCS 2, and SKG I3.⁴⁷ They subsequently analyzed gene mutation rates, protein expression, apoptosis, and cell proliferation in these cell lines in vitro. CRISPR/Cas9 inhibits the interaction between PTPN14 and E7 by mutating PTPN14, inducing downregulation of E6 and E7, associated with inactivation of p53 and RB, leading to programmed cell death and senescence, thereby reducing oncogenic activity. Thus, inhibition of E6 and E7 expression in high-risk HPV using CRISPR/Cas9 provides a precise and effective treatment for HPV infection.

In a compelling suite of investigative trials, the strategic inhibition of the E6 oncogene was found to revitalize the p53 pathway, crucial for tumor suppression. Concurrently, efforts targeting the E7 oncogene were successful in reigniting the retinoblastoma protein (Rb) pathway, another bulwark against tumorigenesis. Zheng et al crafted a precise sgRNA aimed at HPV16-E7, which effectively curtailed E7 expression, augmented levels of functional pRB, and hampered the proliferation of HPV-positive cancer cells.⁴⁸ Extending this line of inquiry, Zhen et al engineered an array of sgRNAs that concurrently besieged both E6 and E7 at the transcriptional level, precipitating a resurgence of p53 and pRB activity.⁴⁹ Jubair et al's vital in vivo research in 2019 deployed these sgRNAs through CRISPR/Cas9 vectors into murine models, subsequently unwinding the sequelae in neoplastic tissues. Results from this study illuminated the potential of such interventions to drive apoptosis and eradicate malignant cells.⁵⁰

In addition to targeting the E6/E7 genes, researchers have also focused on investigating alternative mechanisms of HPV carcinogenesis using CRISPR-Cas9 systems. In 2017, Das D et al demonstrated the significant regulatory role of the cytosolic enzyme SIRT1 in HPV16 replication through CRISPR-Cas9-mediated knockout of the SIRT1 gene.⁵¹ In 2020, the same research group further investigated the regulation of the SIRT1-WRN axis using CRISPR-Cas9 technology. They found that the replication cycle of the virus was dependent on WRN.⁵² In 2023, another research team prepared recombinant lentivirus and TCR-T cells overexpressing transgenic TCR. They employed multicolor flow cytometry (FCM) to detect the expression levels of TCR and CD3 in TCR-T cells, and luciferase activity assay was conducted to evaluate the killing efficiency of TCR-T cells against HPV16-positive SiHa cells. The findings concluded that TCR-T cells lacking endogenous TCR significantly enhanced transgenic TCR expression and exhibited high targeted killing ability towards HPV16-positive cervical cancer SiHa cells, providing an experimental basis for improving clinical efficacy of TCR-T cell therapy.⁵³ Simultaneously, some researchers have initiated investigations into host proteins interacting with E6/E7 gene transcript products. For instance, a study in 2020 revealed that binding between E7 oncoprotein from HPV16/HPV18 and host tumor suppressor PTPN14 repressed differentiation gene expression. Furthermore, experiments demonstrated that CRISPR-Cas9 mutation targeting PTPN14 gene in cervical cancer cells significantly reduced carcinogenic activity induced by HPV virus.⁵⁴ These series of experimental results highlight how knockout of endogenous T-cell receptor (TCR) through CRISPR/Cas9 gene editing technology can substantially enhance specific recognition and killing capabilities against HPV16-positive cervical cancer SiHa cells.

Development and Prospects

With regard to HPV detection, the ideal diagnostic test should accurately and sensitively identify pathogens, while also being affordable, portable, and capable of distinguishing between different pathogen variants, for rapid and cost-effective detection of infectious pathogens or genetic mutations.⁵⁵ CRISPR/Cas and isothermal amplification (NASBA or a combination of the RPA) detection method is likely to enter the laboratory and field in the near future diagnosis. The production and distribution of portable and relatively inexpensive CRISPR diagnostic platforms will greatly improve the treatment of these diseases.¹⁰

Regarding the treatment of HPV, with the increase of HPV vaccination rates the HPV infection rates show a downward trend. At the same time, vaccines such as cervarix, gardasil and Gardasil 9 have also shown high immunogenicity, and almost 100% seroconversion has been achieved. However, the burden of testing and treatment for HPV-related malignancies will remain high in the coming years due to insufficient HPV vaccination coverage and failure to meet herd immunity standards. Therefore, there is an unmet need for effective treatment options for HPV. CRISPR/Cas technology provides a new approach to address this issue, making it possible to detect and treat most cancers caused by HPV from a specific perspective. This therapy has several advantages over conventional approaches: simple design, ease of use, and efficient editing; It provides a promising way for the development of clinical application. Nonetheless, nearly complete CRISPR/ Cas-related gene therapy programs are still in the experimental stage and still subject to off-target effects and other safety and ethical risks. It is expected that with the continuous advancement of CRISPR/Cas technology, most cancers caused by HPV can be effectively treated by gene therapy such as CRISPR/Cas.

At the same time, in terms of clinical application, how to use CRISPR-Cas technology for clinical detection and treatment of HPV also faces many challenges. Off-target effect is the main limiting factor affecting the clinical application of CRISPR technology. At present, the strategies to solve the off-target effects are mainly “predict the off-target site” and “optimize the design strategy of sgRNA and the structure of Cas enzyme”. For example, Tru-RFNs created by TSAI S Q and RAN F A reduce the off-target rate by reducing the single subunit cleavage behavior.^56,57 Vakulskas et al.⁵⁸ found that the RNP complex with the R691A SpCas9 mutant introduced efficient gene editing in human HSPCS, thereby achieving the goal of reducing off-target editing. At the same time, the immunogenicity of Cas protein, accurate delivery of CRISPR/Cas components, viral escape, and the carcinogenicity of CRISPR/Cas system are also great challenges in clinical application,⁵⁹ so more experimental studies are needed.

Footnotes

Acknowledgments

The author declares that the contents of this article have not been published previously.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical Statement

ORCID iD

Minxue Tang

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