CRISPR-Cas genome editing system in the diagnosis and therapy of infection caused by herpes simplex virus type 1 (Orthoherpesviridae: Alphaherpesviridae: Simplexvirus: Simplexvirus humanalpha1)

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Abstract

Herpes simplex virus type 1 (HSV-1), newly named as Simplexvirus humanalpha1 is one of the most common pathogens in the human population, which can cause severe disease, often with fatal outcomes. Diagnostic methods currently in use are specific and sensitive, but time-consuming, require expensive laboratory equipment and highly qualified personnel. Existing therapeutic agents have a number of significant drawbacks. To successfully treat and prevent the spread of the infection, new rapid, easy-to-use, and highly sensitive diagnostic tools and effective therapeutic agents are required. One approach to achieve this goal is CRISPR-based technology.

This review analyzes information obtained from a literature search in the Scopus, Web of Science and MedLine databases on the topics «HSV-1, structure, distribution, life cycle», «new methods for molecular diagnosis of HSV-1-infection», «classification of CRISPR-Cas systems», «nucleic acid amplification methods», «CRISPR-Cas effector proteins», «application of CRISPR-Cas systems in molecular diagnostics of HSV-1-infection», «application of CRISPR-Cas systems in therapy of HSV-1-infection». New approaches of CRISPR using effector proteins Cas12 and Cas13 in the diagnosis of HSV-1 infections are reviewed. The article discusses the progress in the development of CRISPR-Cas-based therapies against HSV-1-infection in vitro and in vivo. CRISPR gene therapy in vivo has a great clinical potential, but its safety and efficacy require further investigation. An analysis of the available data suggests that CRISPR-based technologies offer promising prospects for expanding the arsenal of diagnostic tools and antiviral drugs in the context of current and future outbreaks of viral diseases.

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Introduction

Orthoherpesviridae – a family of DNA viruses, including three subfamilies: Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae. One of the most well-studied representatives of the Alphaherpesvirinae subfamily is herpes simplex virus type 1. The new name of the virus, adopted in 2023, Simplexvirus humanalpha1, has not yet become widely used, so the classic name of the virus, HSV-1, will be used in this article.

HSV-1 is one of the most common pathogens in the human population. According to the World Health Organization (WHO), approximately 70% of people worldwide are infected with HSV-1 [1]. HSV-1 can cause severe diseases, especially in immunocompromised individuals, including herpetic keratitis, encephalitis, neonatal herpes and Alzheimer’s disease [2–7]. Genital herpes is a major cause of genital ulcers, contributes to the transmission of the virus to newborns, causing fatal diseases in them, and significantly increases the risk of contracting the human immunodeficiency virus (HIV-1) [8].

In the treatment of HSV-1 infections, drugs based on nucleotide/nucleoside analogs of nucleic acids (acyclovir, valacyclovir, famciclovir, etc.) are used, which block replication [9], but are unable to eliminate latent forms of the virus. Furthermore, the effectiveness of treating herpesvirus infections is significantly reduced due to the emergence of an increasing number of HSV-1 strains resistant to antiviral drugs. These problems highlight the necessity for the development of new approaches for the prevention and treatment of diseases caused by HSV-1 [10]. Recently, the CRISPR-Cas genome editing technology has been attracting increasing attention from researchers [11]. CRISPR-Cas technology involves creating short guide RNAs that recognize pre-defined nucleic acid sequences, which are then modified by specific proteins within the system – nucleases. CRISPR-Cas systems have been actively used in recent years to develop tools capable of reducing HSV-1 activity and targeting latent viral genes [12, 13]. Thus, the creation and application of the CRISPR-Cas9 system in experiments on infected cell cultures allowed for the complete suppression of HSV-1 replication [14], which opens up prospects for the development of new therapeutic agents. The possibilities of using CRISPR-Cas systems to create new methods for diagnosing herpesvirus infections are also being investigated [15, 16].

This review focuses on the latest advancements in the diagnosis and treatment of HSV-1 infections achieved through the development and application of CRISPR-Cas technology.

HSV-1 life cycle

The viral genome is represented by a linear double-stranded DNA approximately 152 kb in size, potentially encoding about 80 genes. The genome is enclosed within an icosahedral capsid, surrounded by an amorphous layer – the tegument – and an envelope containing viral glycoproteins that are involved in cell attachment and entry [17]. The life cycle of a virus can be divided into several main stages, as shown in Fig. 1 [18–20].

 

Fig. 1. Schematic representation of the life cycle of Simplexvirus humanalpha1 (HSV-1).

1 – attachment and entry into the cell. The gB glycoprotein binds to receptors on the cell surface, mediating primary attachment. gD interacts with HVEM, nectin 1, and sulfated sugars, and the gH/gL complex facilitates viral entry by endocytosis; 2 – transport into the nucleus. After fusion of the viral envelope with the cell membrane, the capsid with some of the tegument proteins moves to the nucleus along microtubules; 3 – initiation of transcription. The tegument protein VP16 is separated from the capsid and, together with cellular factors, activates the immediate early (IE) genes [20]; 4 – cascade gene expression. Immediate early (IE) genes encode factors (ICP4, ICP0, ICP27, ICP22) that regulate transcription, processing, and export of viral mRNA. E genes (early) provide the synthesis of proteins necessary for DNA replication. After DNA replication, L genes (late) are expressed, encoding structural proteins that form the capsid and envelope of the virus [19]; 5 – capsid assembly and DNA packaging; 6 – envelope formation. Capsids receive an envelope in membrane compartments (trans-Golgi, endosomes); 7 – release of virions from the cell. Virions exit the cell by fusion of vesicles containing viral particles with the plasma membrane via exocytosis. The figure shows the viral genes IE, E and L used as targets of the CRISPR-Cas system to suppress HSV-1 infection in the studies discussed in the review [21]. The figure was created using the BioGDP.com software [22].

Рис. 1. Схематическое представление жизненного цикла Simplexvirus humanalpha1 (ВПГ-1).

1 – прикрепление и проникновение в клетку. Гликопротеин gB связывается с рецепторами на поверхности клетки, обеспечивая первичное прикрепление. gD взаимодействует с HVEM, нектином 1 и сульфатированными сахарами, а комплекс gH/gL способствует проникновению вируса путем эндоцитоза; 2 – транспорт в ядро. После слияния оболочки вируса с мембраной клетки капсид с частью тегументных белков перемещается к ядру по микротрубочкам; 3 – запуск транскрипции. Тегументный белок VP16 отделяется от капсида и вместе с клеточными факторами активирует сверхранние (IE) гены [20]; 4 – каскадная экспрессия генов. Сверхранние IE-гены кодируют факторы (ICP4, ICP0, ICP27, ICP22), регулирующие транскрипцию, обработку и экспорт вирусных мРНК. E-гены (ранние) обеспечивают синтез белков, необходимых для репликации ДНК. После репликации ДНК экспрессируются L-гены (поздние), которые кодируют структурные белки, формирующие капсид и оболочку вируса [19]; 5 – сборка капсидов и упаковка ДНК; 6 – формирование оболочки. Капсиды получают оболочку в мембранных компартментах (транс-Гольджи, эндосомы); 7 – выход вирионов из клетки. Слияние везикул, содержащих вирусные частицы, с плазматической мембраной обеспечивает выход вирионов путем экзоцитоза.

На рисунке обозначены вирусные гены IE, E и L, использованные в качестве мишеней системы CRISPR-Cas в работах, рассмотренных в обзоре [21]. Рисунок составлен с помощью программы BioGDP.com [22].

 

Transcription and replication of viral DNA occur within the nucleus of the infected cell. Viral gene transcription is strictly implemented according to a cascade principle: first immediate early (IE), then early (E), and finally late (L) genes. Viral DNA replication proceeds via a rolling circle mechanism. Replication involves the proteins pUL9 (origin-binding protein), ICP8 (single-stranded DNA-binding protein), pUL30/pUL42 (DNA polymerase complex), and pUL5/pUL8/pUL52 (helicase-primase complex). Additional (auxiliary) proteins are also involved in the synthesis of viral DNA: pUL12 (viral 5’→3’ alkaline exonuclease), pUL23 (thymidine kinase), a ribonucleotide reductase consisting of two homodimeric subunits, R1 (pUL39) and R2 (pUL40) and others [23].

Recombination during HSV-1 replication can begin at sites of single-strand or double-strand DNA breaks. The appearance of single-strand breaks hinders normal replication and provides additional opportunity for recombination events. Double-strand DNA breaks trigger the isomerization of the HSV-1 genome, with recombination events particularly affecting the inverted repeats at the ends of the genome [24]. Such intergenomic rearrangements lead to the emergence of new strains and contribute to the genetic diversity of HSV-1 in nature [17, 24].

During replication, viral DNA can form branched structures resembling Y- and X-junctions. Y-junctions reflect replication forks, while X-junctions can be recombination intermediates or alternative replication structures [17]. Although cellular factors are also involved in these processes, HSV-1 encodes its own recombinase, consisting of two viral proteins: the alkaline nuclease pUL12 and the single-stranded DNA-binding protein ICP8 [24, 25]. Together, they catalyze chain exchange, which allows for the rearrangement of genome segments [17]. Capsid assembly and packaging occur in the nucleus. Then, nucleocapsids acquire an envelope in the membranes of the Golgi apparatus and in endosomes and exit the cell by fusing transport vesicles with the plasma membrane. Thus, for assembly, egress, and cell-to-cell spread, HSV-1 utilizes exosome biogenesis pathways [26].

After the initial infection, HSV-1 enters sensory neurons in epithelial cells and becomes latent, primarily in the trigeminal ganglia [27]. Here, the expression of viral proteins is severely limited, which is supported by low levels of VP16 and ICP0, as well as epigenetic regulatory mechanisms involving cellular factors IFI16 and PML [28]. However, under stressful conditions (UV irradiation, neuronal damage, or hormonal changes), signaling pathways (JNK, Egr-1/Sp1) are activated, the synthesis of VP16 and ICP0 increases, and the virus begins to re-express its own genes, leading to reactivation and the formation of new infectious virions [29, 30].

CRISPR-Сas system

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated proteins (Cas) provide bacteria and archaea with adaptive immunity against viruses, plasmids, and other foreign genetic elements [11]. This defense system is based on small RNAs to detect and suppress foreign nucleic acids. CRISPR/Cas systems consist of Cas genes organized into operons and a CRISPR array. It consists of unique sequences (spacers) interspersed with identical repeats. Spacers usually correspond to fragments of viral genomes that previously infected the cell.

CRISPR-Cas-mediated defense occurs in three stages. In the first stage, called adaptation, bacteria and archaea carrying one or more CRISPR loci respond to viral and plasmid genetic material by integrating short fragments of foreign sequence (protospacers) into the host chromosome at the proximal end of the CRISPR array. In the second stage, called expression, the entire CRISPR locus is transcribed, forming a long molecule – the precursor CRISPR RNA (pre-crRNA) – which is then enzymatically cleaved into short crRNAs containing a repeat fragment and a spacer. Transcription of Cas genes also occurs. In the third stage, called interference, Cas proteins, by combining with crRNA (and sometimes tracrRNA), form a ribonucleoprotein complex that finds and destroys complementary viral or plasmid nucleic acids [11].

CRISPR-Cas system classification

Several CRISPR-Cas systems have been discovered in nature, which are divided into numerous categories defined by 2 classes, 6 types and 33 subtypes. Currently, all CRISPR-based technologies for detecting viral nucleic acids and their modifications utilize class 2 Cas effector proteins. Class 2 systems are easy to implement in mammalian systems because a single multi-domain effector protein is responsible for cleaving nucleic acids [31]. This class is further subdivided into types II, V, or VI depending on the Cas effector protein, which is Cas9, Cas12, and Cas13, respectively (Fig. 2). Each Cas effector protein has a different domain composition and a specific mechanism of action [32].

 

Fig. 2. Class 2 CRISPR-Cas effector proteins.

The figure is adapted from [32] using the BioGDP.com software [22].

Рис. 2. Эффекторные белки CRISPR-Cas класса 2.

Рисунок адаптирован на основе работы [32] с помощью программы BioGDP.com [22].

 

Type II systems (Cas9) are the most well-studied. Cas9 cleaves target DNA to create blunt ends using its RuvC and HNH nuclease domains, employing a relatively small guide RNA spacer to recognize the target. This system is widely used in genome editing technologies, which are frequently employed in both basic research and applied fields such as biotechnology, food industry, and medicine.

Type V (Cas12) systems contain smaller protein effectors than type II systems, which cleave the DNA target using a single RuvC domain. Unlike Cas9, Cas12 creates single-stranded overhangs when cutting DNA. This increases the accuracy of gene editing because single-stranded overhangs are better suited for repairing double-strand DNA breaks through homologous recombination. As a result, the insertion and modification delegations become more controlled.

Type VI systems (Cas13) were characterized relatively recently, and their target is single-stranded RNA, meaning the Cas13 effector is an RNA-guided RNA-specific nuclease. CRISPR-Cas13d systems are a promising tool for knocking down specific RNA sequences without altering the genome sequence, offering a safer alternative in situations where inherited off-target DNA changes are undesirable [33].

Combination of CRISPR-Cas with isothermal amplification for the diagnosis of viral infections

The combination of CRISPR technology with isothermal amplification methods (IAT), which are simple and versatile, allows it to be used both in the field and in laboratory settings [34, 35]. The main IAT methods include loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and recombinase-aided amplification (RAA) [36]. LAMP and RPA do not require a thermal cycler and can be performed at a single temperature, making them convenient for point-of-care testing [37–40].

CRISPR-based nucleic acid detection technologies

Nucleic acid detection technologies are discussed because viral genomes are represented by DNA or RNA molecules of varying structures. Early technologies used the canonical Cas9 protein from type II CRISPR-Cas systems [41]. The discovery of the collateral cleavage activity of Cas12 and Cas13 proteins was a huge step toward the development of CRISPR-based molecular diagnostics. One advantage of Cas12 and Cas13 proteins compared to Cas9 for use in genetic engineering is their smaller size and lower sensitivity to nucleotide mismatches between the target DNA and crRNA [31]. The study of the collateral cleavage activity of Cas12 and Cas13 has led to the development of a range of convenient CRISPR-based nucleic acid detection methods that can be used in the field and are also scalable. CRISPR-based detection technologies developed to date utilize various sample processing and amplification methods, different Cas effector proteins, and different ways to assess results; they can be applied to a wide range of both DNA and RNA viruses, highlighting the versatility of these systems for detecting viral nucleic acids [42]. Fig. 3 shows a generalized scheme of various nucleic acid detection methods [32]. The discovery of RNA cleavage using the CRISPR-Cas13a system has revolutionized the field of virus detection. In 2017, J.S. Gootenberg et al. developed the SHERLOCK (Specific High Sensitivity Enzymatic Reporter unLOCKing) virus detection technology based on the CRISPR/Cas13 system. This platform is capable of detecting both RNA and DNA, and is characterized by its versatility, ease of use, and high sensitivity [43]. The RNA target is either detected directly in one step or in combination with RT-RPA isothermal amplification in two steps. Cas13 binds to target RNA sequences, and detection of the amplified target is achieved by introducing a synthetic single-stranded RNA molecule surrounded by a quencher and attached to a fluorescent dye. Due to the non-specific cleavage property of Cas13, a fluorescent signal is generated, which is detected by a reader [44]. The SHERLOCK system has been adapted for the diagnosis of several viruses, such as the Dengue virus and the Zika virus [44–47]. Cas13a-based assays enable rapid detection of hemorrhagic fever viruses, including Lassa and Ebola, in the field without prior amplification or transcription [48]. However, it should be noted that none of the variants of such tests have yet been approved by the WHO.

 

Fig. 3. Experimental CRISPR/Cas12- or CRISPR/Cas13-based viral nucleic acid detection methods.

Most CRISPR-based detection methods require sample processing, Cas12- or Cas13-mediated amplification and detection with a defined readout. Sample processing can be performed by filtration or magnetic extraction, or various chemical and/or thermal inactivation approaches. Amplification is performed using PCR or isothermal methods such as RPA and LAMP. Detection is mediated by Cas12 and Cas13 and the results are read fluorescently or visually. Figure adapted from [32].

Рис. 3. Экспериментальные методы обнаружения вирусных нуклеиновых кислот на основе CRISPR/Cas12 или CRISPR/Cas13.

Большинство методов обнаружения вирусов на основе CRISPR требуют предварительной обработки образцов, амплификации и детекции, опосредованной Cas12 или Cas13, с соответствующим анализом результатов. Обработка образцов может быть выполнена с помощью фильтрационной или магнитной экстракции или различных химических и/или термических подходов инактивации. Амплификация выполняется с использованием полимеразной цепной реакции или изотермических методов, таких как RPA и LAMP. Обнаружение – с помощью Cas12 и Cas13, анализ результатов – методом иммунохроматографии с визуальной оценкой или количественным анализом интенсивности флуоресценции. Рисунок адаптирован на основе работы [32].

 

CRISPR-Cas13a assays, including HUDSON-SHERLOCK and SHERLOCKv2, played a significant role in the rapid identification of respiratory viruses, thus contributing to the fight against the SARS-CoV-2 pandemic [49–51]. This technology is unique for the rapid detection of disease pathogens, especially in differentiating different strains of viruses and other pathogens, and plays a key role in protecting human health [36, 44, 52].

The Cas12 protein has been used to detect nucleic acids in technologies such as DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter), which targets DNA endonuclease [53], and HOLMES (an one-HOur Low-cost Multipurpose highly Efficient System) [54, 55]. The use of these methods eliminated the need for intracellular transcription after amplification, as Cas12 can detect the direct amplification product (double-stranded DNA). Cas12 activity in cleaving the DNA double helix was triggered by recognizing the viral DNA target, and the introduced single-stranded DNA suppressed the fluorescent reporter molecule, leading to the quantitative cleavage of the probe [53, 56]. In these two Cas12-based platforms, different amplification approaches were used: RPA in DETECTR and polymerase chain reaction (PCR) in HOLMES [32]. DETECTR was first applied to detect human papillomavirus types 16 and 18 (HPV16 and HPV18) DNA. A comparative analysis with quantitative PCR showed a 92% match in DNA detection [56]. In a study by L. Yin et al., a new platform based on CRISPR-Cas12a technology was developed for rapid, highly sensitive, and specific detection and genotyping of 14 high-risk HPV types. This platform also enabled multiplex detection of 14 high-risk HPV types in a single tube [57]. Cas12-based diagnostics play an important role in the direct detection of DNA viruses without RNA transcription [45].

In 2022, X. Wu et al. introduced a digital method called WS-RADICA (Warm-Start RApid DIgital Crispr Approach) for nucleic acid quantification, based on the CRISPR-Cas12b system [58]. The authors showed that using a hot start allows the test to be performed at room temperature. Using the WS-RADICA test, only 1 copy/µL of SARS-CoV-2 RNA was detected in 40 minutes (qualitative detection) or 60 minutes (quantitative detection). The WS-RADICA method can be easily adapted to evaluate test results from various digital devices. The authors concluded that due to its speed, sensitivity, quantification ability, and resistance to inhibitors, WS-RADICA is a promising method for various diagnostic applications requiring nucleic acid quantification.

The developed technologies, based on combinations of CRISPR-Cas with isothermal amplification, have ushered in a new era in molecular diagnostics, providing portable and highly sensitive diagnostic tools suitable for detecting emerging infectious and non-infectious diseases.

Application of the CRISPR/Cas system for diagnosing HSV-1 infections

Currently, the real-time PCR method is widely used in practical medicine for detecting HSV-1. However, the available domestic reagent kits for detecting HSV-1/2 DNA in clinical samples by PCR are not designed for absolute quantitative analysis. English-language literature contains data on determining the quantity of HSV-1 DNA, including in-house PCR, with varying analytical sensitivity. To improve the quality of HSV-1 infections diagnosis, it is necessary to refine traditional methods and develop new approaches, including those based on CRISPR-Cas technology.

X. Yin et al. developed the HSV-SHERLOCK test for the detection and genotyping of HSV, based on CRISPR RNA (crRNA) that targets 8 different regions of the HSV-1/2 UL30 gene [59]. Fig. 4 shows the testing stages using HSV-SHERLOCK. The authors reported high sensitivity (96.15% for HSV-1) and 100% specificity compared to a commercial real-time PCR kit when testing 194 clinical samples. Notably, the analysis can detect 1 copy of viral DNA in 1 μL and differentiate between HSV-1 and HSV-2. Furthermore, the test results can be evaluated both by the appearance of color (colorimetrically, without the use of instruments) and by computer data processing. These results demonstrate the high efficiency of a CRISPR-based test for diagnosing HSV infections that does not require expensive equipment, and the potential of HSV-SHERLOCK for testing in resource-limited settings.

 

Fig. 4. Schematic representation of the steps of the HSV-SHERLOCK assay for the detection of HSV-1.

Clinical samples were incubated at 100 °C for 10 min for DNA extraction; the target gene of HSV-1 is preamplified with a set of universal primers by RPA in a single tube with extracted DNA as the input; RPA products were further transferred into the separate tubes containing target-specific crRNA for Cas13a detection; the fluorescence measurement results were recorded using a microplate reader.

The figure is adapted from the article [59] using the BioGDP.com software [22].

Рис. 4. Схематическое представление этапов HSV-SHERLOCK анализа для обнаружения ВПГ-1.

Клинические образцы инкубировали при 100 °C в течение 10 мин для выделения ДНК; затем ген-мишень ВПГ-1 предварительно синтезировали с применением набора универсальных праймеров с помощью RPA в одной пробирке с экстрагированной ДНК из исходного материала; продукты RPA переносили в отдельные пробирки, содержащие мишень-специфичную crРНК, для детекции с использованием Cas13а; результаты флуоресцентного анализа обрабатывалия и записывали с помощью компьютерных программ.

Рисунок адаптирован на основе работы [59] с помощью программы BioGDP.com [22].

 

B. Dou et al. developed a novel photoelectrochemical method for detecting HSV-1 using multiple potential step chronoamperometry (MUSCA) based on CRISPR-Cas12a. MUSCA allows for real-time tracking and monitoring of changes in the concentration of the analyte, observing all dynamic changes in the current. This unique feature allows for the transformation of certain biological processes, such as the rapid release and reaction of electroactive molecules. It has been shown that the MUSCA method allows for obtaining strong photoelectrochemical signals (PEC) on an electrode upon illumination. This strategy enabled sensitive virus detection by monitoring changes in MUSCA-FES signals. The MUSCA-FES strategy was successfully applied for the detection of HSV-1 in human blood serum samples [15].

In 2023, M. Huang et al. proposed a new method for diagnosing keratitis caused by HSV-1. It incorporates CRISPR-Cas12a technology combined with LAMP and gold nanoparticles. Fig. 5 shows a schematic diagram of the colorimetric analysis for detecting HSV-1 DNA using this method. The LAMP reaction was used to amplify HSV-1 DNA extracted from clinical tear fluid samples. The Cas12a-crRNA system recognized the HSV-1 gB protein gene in the presence of gold nanoparticles (AuNP) and cleaved single-stranded DNA (ssDNA), releasing the gold nanoparticles. The test result was visible to the naked eye and was evaluated calorimetrically. The authors demonstrated the high sensitivity and specificity of the method, as well as its speed of execution and the lack of necessity for complex equipment [60].

 

Fig. 5. Schematic representation of the CRISPR/Cas12-based colorimetric assay for the detection of HSV-1.

Figure adapted from [60] using BioGDP.com software [22].

Рис. 5. Схематическое представление колориметрического анализа на основе CRISPR/Cas12 для обнаружения ВПГ-1.

Рисунок адаптирован на основе работы [60] с помощью программы BioGDP.com [22].

 

Figure adapted from [60] using BioGDP.com software [22].

Application of the CRISPR/Cas system for the therapy of HSV-1 infection

The first cell therapy product derived using CRISPR-Cas9 gene editing technology, Casgevy, has recently been approved for the treatment of severe sickle cell disease and transfusion-dependent β-thalassemia1. This important event is inspiring researchers to improve genome editing technology for application in various fields of medicine, including the treatment of infectious diseases of viral etiology.

Experiments to investigate the potential use of the CRISPR-Cas system for treating diseases caused by HSV-1 were conducted shortly after the discovery of the CRISPR-Cas genome editing technology. Initially, in vitro models – cultured cells infected with HSV-1 – showed that the CRISPR/Cas system, targeting specific viral genes, is capable of suppressing viral replication. It has been established that approximately 30 viral genes or their combinations, described in detail in study [21] and indicated in Fig. 1, could in principle become targets for the CRISPR-Cas system for the treatment of human herpesvirus diseases. Interestingly, suppression of infection in cells was observed when viral genes active at different stages of the HSV-1 life cycle were used as targets, as shown in Fig. 1, although the effectiveness varied [23]. It should be noted that different compositions of the CRISPR-Cas system components and various methods of their delivery were used.

In addition to conventional 2D cell cultures, A. Bellizzi et al. [61] used 3D cultures: they induced differentiation into neurons in human pluripotent stem cells and obtained three-dimensional «cerebral organoids» (COs). Two very early genes of HSV-1 (ICP0 and ICP27) were chosen as targets for the CRISPR-SaCas9 system, and adeno-associated virus AAV2 was used as the vector. Transduction of Vero cells with AAV2-SaCas9-ICP0 or AAV2-SaCas9-ICP27 resulted in a significant reduction in the quantity and infectious activity of HSV-1. The action of the AAV2-SaCas9-ICP0 or AAV2-SaCas9-ICP27 systems was also analyzed in the DRG after establishing the latent form of infection and subsequent reactivation of HSV-1. A 2.5–5.0-fold decrease was observed in the number of organelles in which reactivation occurred, as well as an approximately 300-fold reduction in the number of HSV-1 DNA copies. The data obtained showed that the CRISPR-SaCas9 system targeting the ICP0 and ICP27 genes effectively suppresses HSV-1 reactivation in DRG neurons. Furthermore, it has been shown that targeting multiple different sites within the HSV-1 very early genes using CRISPR-SaCas9 is capable of inducing large deletions in the target genes, suggesting that this technology could be considered a tool for eliminating HSV-1 DNA from latently infected cells.

The successful suppression of HSV-1 activity in cells in vitro allowed for the transition to evaluating the effectiveness of HSV-1 genome editing in vivo models. One of them is herpes simplex encephalitis (HSE) in mice. Herpes simplex virus type 1 (HSV-1) encephalitis is characterized by high mortality and is the most common form of viral encephalitis in humans worldwide [62]. Antiviral drugs – acyclovir and its analogs – can reduce mortality from encephalitis from 75% to 20%, [4], and there is hope that the safety and effectiveness of treatment can be further improved, including through new CRISPR/Cas technologies.

M. Ying et al. [63] conducted experiments to evaluate the effect of HSV-1 genome editing in a mouse model of intracerebral infection. CRISPR/Cas system components were introduced into animals as part of a lentiviral vector before or after infection. They studied the replication and spread of the virus in the brains of mice. To achieve this, they developed a strategy called CLEAR (coordinated lifecycle elimination against viral replication), which aims to suppress the HSV-1 proteins VP16, ICP27, ICP4, and gD. The study results showed that lentiviruses containing CRISPR-Cas9-gRNA, introduced 5 days before infection, effectively blocked HSV-1 replication in the brains of mice. Significant inhibition of viral proliferation was also observed using the CLEAR system the day after HSV-1 infection. The authors concluded that the CLEAR strategy was effective both prophylactically and therapeutically, protecting mice from HSV-1 brain infection. Further research will show the prospects for using the developed strategy to treat HSE.

Corneal inflammation caused by external HSV-1 infection or reactivation of the latent virus – herpetic stromal keratitis (HSK) – is a leading cause of blindness worldwide [64]. In study [65] the possibility of using CRISPR-Cas9 to treat HSV-1 keratitis was investigated in an HSV-1 mouse model. The authors developed lentiviral particles containing SpCas9 mRNA targeting two HSV-1 genes – UL8 and UL29, which are involved in viral replication, and named them HELP (HSV-1-erasing lentiviral particle). The study results showed that a single injection of HELP into the cornea significantly reduced the level of HSV-1 in the cornea and in the trigeminal ganglia (TG) of mice.

N. Amrani et al. used CRISPR-Cas9 gene editing technology on a model of latent herpesviral keratitis in rabbits [66]. Fig. 6 shows a schematic representation of the experiment conducted. Rabbits were infected with HSV-1 by corneal scarification. After 4 weeks, latency was established in the TG ganglia, and no infectious virus was detected in eye swabs. Reactivation was induced by physicochemical effect on the eye’s cornea. The genes encoding two herpes simplex virus 1 (HSV-1) immediate-early proteins – ICP0 and ICP27 – were chosen as targets for CRISPR-Cas9. The authors considered that ICP0 overlaps with the LAT locus, and ICP27 is located in an adjacent region, which is important for the effectiveness of targeting latent HSV-1. Furthermore, ICP0 is duplicated in the HSV-1 genome. Therefore, using two gRNAs can lead to three double-strand DNA breaks and various deletions in the latent HSV-1 genome, preventing viral replication. The study used SaCas9 nuclease and two chimeric gRNAs targeting the selected genes, which were delivered using a single adeno-associated viral vector (AAV). It was shown that introducing the created system through corneal scarification reduced viral shedding by more than 50%. Intravenous administration of the same AAV9-SaCas9 vector completely suppressed viral shedding in 11/12 (92%) of the treated eyes. Furthermore, a decrease in the levels of viral DNA and LAT RNA was observed in the TG of treated rabbits. The results obtained confirm the effectiveness of single-dose comprehensive gene editing using the AAV9-CRISPR-Cas9 system as a safe and effective treatment strategy for HSV-1 induced keratitis.

 

Fig. 6. Schematic representation of the design of experimental HSV-1 ocular infection in rabbits and treatment with CRISPR-Cas9 components delivered by an AAV9 vector – AAV9-CRISPR-Cas9.

To reactivate the virus from latency, a stress stimulus was applied in which each rabbit’s eye received transcorneal iontophoresis of epinephrine (TCIE), a procedure in which a small electric current drives the uptake of epinephrine into the eye, for 3 consecutive days. Adapted from [66] using BioGDP.com software [22].

Рис. 6. Схематическое изображение дизайна экспериментальной глазной инфекции у кроликов, вызванной ВПГ-1, и лечения компонентами CRISPR-Cas9, доставляемыми с помощью вектора AAV9 – AAV9-CRISPR-Cas9.

Для реактивации использовали транскорнеальный ионофорез эпинефрина (TCIE) – процедуру, при которой небольшой электрический ток обеспечивает поступление эпинефрина в глаз в течение 3 сут подряд. Адаптировано из [66] с помощью программы BioGDP.com [22].

 

Active HSV infection in the neonatal period affects approximately 14,000 newborns worldwide annually, leading to severe nervous system disorders and high mortality rates. The impact of subclinical HSV infection in the neonatal period on the adult CNS is poorly understood. A.J. Dutton et al. [67] reported that newborn mice intranasally infected with low doses of HSV did not show clinical signs of acute infection, but viral genome and latency-associated transcripts (LAT) were detected in the CNS 6 months later. Adult animals showed a decrease in their ability to perform a range of cognitive and memory tests. The authors pointed out a link between viral infection and nervous system dysfunction and proposed a new model for studying neurodegenerative diseases like Alzheimer’s disease. The proposed model opens up the possibility of using CRISPR-Cas9 technology in the neonatal period to prevent CNS disorders later in life.

The fundamental ability of the CRISPR-Cas9 system to suppress HSV-1 infection in the CNS was demonstrated in a study by R. de Sousa et al. [3]. Mice were infected with HSV-1 directly into the eyeball, and 24 hours later, the CRISPR-Cas9 complex was administered as a plasmid in eye drops without the use of delivery agents such as viral vectors or nanoparticles. The target of CRISPR-Cas9 was the HSV-1 UL39 gene, which encodes the large subunit of viral ribonucleotide reductase, essential for DNA synthesis, particularly in non-replicating cells like neurons. Viral load analysis showed that on day 7 after infection, the concentration of viral DNA in brain tissues decreased by approximately 300 times (108–105 copies/mL, p < 0.01) in the group receiving CRISPR/Cas9, while no signs of histopathology or brain inflammation were observed in the animals. These results in an in vivo HSV-1 eye infection model suggest that the use of CRISPR/Cas9 targeting UL39 holds promising therapeutic potential for protecting the CNS from neuroinfections.

A. Wei et al. [68] used lentiviral HELP particles, previously developed to treat 3 patients with severe HSK during a clinical trial (NCT04560790) [65]. The HELP drug, targeting the HSV-1 UL8 and UL29 genes, was administered simultaneously with the corneal transplant. Lentiviral particles were injected into the recipient’s graft bed at 6–8 locations. As a result, all 3 patients remained free of viral infection recurrence for 18 months after treatment. However, the authors noted that there are risks of accidental integration into the host genome when using lentiviral vectors. Furthermore, it is unclear to what extent the observed effect was contributed to by the simultaneous corneal transplantation.

For a more detailed analysis of the HELP system effect, Chinese researchers are continuing their work within the Phase I clinical trials, the aim of which is to evaluate the safety, tolerability, and efficacy of the drug in patients with stromal keratitis caused by HSV-12. The HELP-based drug is called BD111 and is a lentiviral particle that is the active pharmaceutical ingredient, delivering gRNA and SpCas9 mRNA expression cassettes. The mechanism of action is based on CRISPR/Cas9 technology. The study includes 4 groups (16 patients) divided to determine the optimal dose of the drug, and one group as a positive control, which will receive therapy with three approved antiviral medications. Positive treatment outcomes will determine the optimal dose of the drug, its safety and effectiveness.

Conclusion

CRISPR-Cas systems, initially discovered as defense mechanisms in bacteria and archaea against the introduction of foreign elements, have demonstrated their significance in molecular research of metabolic processes and pathological conditions in animals and humans in recent years. One important experimental direction is the creation of CRISPR-Cas systems for the diagnosis of viral infections. New technological developments in HSV-1 detection based on CRISPR-Cas platforms such as SHERLOCK, DETECTR, WS-RADICA and MUSCA-FES show significant potential. Even at this stage, they exhibit high specificity and sensitivity, comparable to or exceeding the gold standard – real-time PCR.

The second important direction is the use of CRISPR-Cas systems in the therapy of diseases caused by HSV-1. Studies conducted on cell cultures and animal models have shown the effectiveness of suppressing active infection and the potential for eliminating latent viruses. Clinical trials of a new treatment method for stromal herpetic keratitis using CRISPR-Cas technology have shown promising results, opening up prospects for safe and effective treatment of diseases caused by HSV-1 and other viral agents.

To implement CRISPR-Cas technology in clinical practice, a number of important issues need to be addressed, including: eliminating off-target effects; developing effective methods for delivering system components; preventing potential immune reactions; and ensuring genomic safety. Addressing these challenges through the further development of genome editing technologies opens up prospects for controlling HSV-1 infection due to the dual ability of CRISPR-Cas systems to diagnose and treat.

1 FDA Approves First Gene Therapies to Treat Patients with Sickle Cell Disease. Available at: www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapies-treat-patients-sickle-cell-disease

2 A Study of the Safety, Tolerability and Prelinminary Efficacy of BD111 in Herpes Simplex Virus Type I Stromal Keratitis. Available at: https:// clinicaltrials.gov/study/NCT06474416

×

About the authors

Natalia A. Demidova

Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: ailande@yandex.ru
ORCID iD: 0000-0003-1961-9789

Researcher of the Laboratory of Cell Engineering

Russian Federation, 123098, Moscow

Regina R. Klimova

Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: regi.K@mail.ru
ORCID iD: 0000-0002-4147-8444

PhD (Biology), Senior Researcher of the Laboratory of Cell Engineering

Russian Federation, 123098, Moscow

Alla A. Kushch

Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: vitallku@mail.ru
ORCID iD: 0000-0002-3396-5533

D.Sci. (Biology), Professor, Chief Researcher of the Laboratory of Cell Engineering

Russian Federation, 123098, Moscow

Dmitry S. Karpov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: aleom@yandex.ru
ORCID iD: 0000-0001-5203-0787

PhD (Biology), Leading Researcher

Russian Federation, 119991, Moscow

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2. Fig. 1. Schematic representation of the life cycle of Simplexvirus humanalpha1 (HSV-1). 1 – attachment and entry into the cell. The gB glycoprotein binds to receptors on the cell surface, mediating primary attachment. gD interacts with HVEM, nectin 1, and sulfated sugars, and the gH/gL complex facilitates viral entry by endocytosis; 2 – transport into the nucleus. After fusion of the viral envelope with the cell membrane, the capsid with some of the tegument proteins moves to the nucleus along microtubules; 3 – initiation of transcription. The tegument protein VP16 is separated from the capsid and, together with cellular factors, activates the immediate early (IE) genes [20]; 4 – cascade gene expression. Immediate early (IE) genes encode factors (ICP4, ICP0, ICP27, ICP22) that regulate transcription, processing, and export of viral mRNA. E genes (early) provide the synthesis of proteins necessary for DNA replication. After DNA replication, L genes (late) are expressed, encoding structural proteins that form the capsid and envelope of the virus [19]; 5 – capsid assembly and DNA packaging; 6 – envelope formation. Capsids receive an envelope in membrane compartments (trans-Golgi, endosomes); 7 – release of virions from the cell. Virions exit the cell by fusion of vesicles containing viral particles with the plasma membrane via exocytosis. The figure shows the viral genes IE, E and L used as targets of the CRISPR-Cas system to suppress HSV-1 infection in the studies discussed in the review [21]. The figure was created using the BioGDP.com software [22].

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3. Fig. 2. Class 2 CRISPR-Cas effector proteins. The figure is adapted from [32] using the BioGDP.com software [22].

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4. Fig. 3. Experimental CRISPR/Cas12- or CRISPR/Cas13-based viral nucleic acid detection methods. Most CRISPR-based detection methods require sample processing, Cas12- or Cas13-mediated amplification and detection with a defined readout. Sample processing can be performed by filtration or magnetic extraction, or various chemical and/or thermal inactivation approaches. Amplification is performed using PCR or isothermal methods such as RPA and LAMP. Detection is mediated by Cas12 and Cas13 and the results are read fluorescently or visually. Figure adapted from [32].

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5. Fig. 4. Schematic representation of the steps of the HSV-SHERLOCK assay for the detection of HSV-1. Clinical samples were incubated at 100 °C for 10 min for DNA extraction; the target gene of HSV-1 is preamplified with a set of universal primers by RPA in a single tube with extracted DNA as the input; RPA products were further transferred into the separate tubes containing target-specific crRNA for Cas13a detection; the fluorescence measurement results were recorded using a microplate reader. The figure is adapted from the article [59] using the BioGDP.com software [22].

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6. Fig. 5. Schematic representation of the CRISPR/Cas12-based colorimetric assay for the detection of HSV-1. Figure adapted from [60] using BioGDP.com software [22].

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7. Fig. 6. Schematic representation of the design of experimental HSV-1 ocular infection in rabbits and treatment with CRISPR-Cas9 components delivered by an AAV9 vector – AAV9-CRISPR-Cas9. To reactivate the virus from latency, a stress stimulus was applied in which each rabbit’s eye received transcorneal iontophoresis of epinephrine (TCIE), a procedure in which a small electric current drives the uptake of epinephrine into the eye, for 3 consecutive days. Adapted from [66] using BioGDP.com software [22].

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