CRISPR–Cas system to discover host-virus interactions in Flaviviridae

The Flaviviridae family encompasses a large group of single-stranded, positive-sense RNA viruses. Four genera belong to this family: Flavivirus, Pestivirus, Hepacivirus, and Pegivirus. Some members of the Hepacivirus and Flavivirus genera are responsible for several important human diseases [1]. Hepatitis C virus (HCV) belongs to the Hepacivirus genus, which differs in many aspects compared to the members of the Flavivirus genus, including the transmission route or the course of infection [2]. The main transmission path for most flaviviruses is through arthropod vectors and includes important pathogens such as the Zika virus (ZIKV) [3]. Zika virus infections in pregnant women have been associated with congenital microcephaly and other developmental defects in infants. The aforementioned traits of Zika virus has attracted the attention of the medical community worldwide [4‐6]. Dengue virus (DENV), which causes approximately 100 million symptomatic infections annually, is another cause of infectious diseases inflicted by flaviviruses [7]. Yellow fever virus (YFV) is another member of the Flavivirus genus and is known to be a cause of hemorrhagic fever. It remains prevalent in sub-Saharan Africa and South America, in spite of the availability of a highly effective live-attenuated vaccine against it [8, 9]. Among people infected with West Nile virus (WNV), only about 20% present the symptoms of West Nile fever (WNF). Less than 1% of the infected individuals develop a neuroinvasive disease characterized by encephalitis, meningitis, and flaccid paralysis [10‐12]. So far, no specific or potent antiviral treatments are available against ZIKV, DENV, and WNV infections. Outbreaks still occur despite licensed vaccines against several members of the Flaviviridae family, including DENV, YFV, Japanese encephalitis virus (JEV), and Tick-borne encephalitis virus (TBEV), emphasizing the challenges and flaws in implementing effective vaccination programs [13]. Viruses are obligate pathogens, dependent on their host to complete their replication cycle. Viruses utilize cellular receptors to enter the host and hijack cellular functions and pathways to replicate, assemble and release new virus particles; hence, identifying the cellular factors that promote or restrict virus replication will reveal the fundamental characteristics of host-virus interaction. This, in turn, could lead to the development of target-specific antiviral drugs in the future [14]. Genomic approaches are increasingly being utilized to identify viral pathogenesis mechanisms and study host-viral interactions. Several genetic screening technologies, such as RNA interference (RNAi), haploid embryonic stem cells, and clustered regularly interspaced short palindromic repeats (CRISPR), have proven to be powerful means for examining viral lifecycles [15]. However, the CRISPR/Cas technology as an efficient tool for genomic engineering has overcome the limitations of other competing technologies. Furthermore, this system has been engineered to effectively induce knockout mutations in a wide range of cell types. The expansion of CRISPR/Cas9 screening libraries allows all known genes from any species to be targeted, including a pool of guide RNAs to target a vast variety of genes. Either way, gene knockouts or the activation of gene expression can be achieved [16, 17]. The present review aims to elucidate the basic principles and types of different CRISPR screens, and their use in novel anti-viral approaches. Therefore, we have also provided a comprehensive overview of the recent discoveries about virus-host interactions which have been achieved using CRISPR screens. Lastly, we have described the currently available CRISPR-Cas antiviral strategies against the Flaviviridae family as one of the main groups of lethal viral infections.

CRISPR/Cas system is an adaptive immune mechanism protecting the bacteria against invading viruses and plasmids [18, 19]. Bacterial CRISPR loci consist of a Cas operon and a repeat-spacer array. This defensive process can be divided into three stages. The acquisition step involves the integration of foreign nucleic acids into a CRISPR array as new CRISPR spacers separated by repeat sequences found adjacent to the CRISPR-associated (Cas) genes, which encode Cas ribonucleases. This step creates a memory of the foreign genetic components [20]. In the second step (expression), the CRISPR array must be transcribed into a pre-CRISPR RNA transcript (pre-crRNA), then processed. The outcome of this step at this stage is finally a mature crRNA [21, 22]. Additionally, a transactivating RNA (tracrRNA) is also encoded by the CRISPR locus, which has complementarity properties to the repeat areas of crRNA transcripts [21]. Subsequently, in the third stage (i.e. interference), through the binding of complementary repeat region sequences, the crRNA-tracrRNA hybrid is formed. Finally, Cas nuclease is guided to the complementary DNA sequences using this RNA hybrid, targeting and cleaving the nucleic acids derived from the invading viruses and other genetic elements [23].

The CRISPR/Cas systems are divided into two categories according to their effector molecules: multi-subunit effector molecules in Class 1 and a single effector molecule in Class 2. The first class can be subdivided into three types (I, III, and IV), and Class 2 consists of types II, V, and VI [24, 25]. Specifically, the CRISPR/Cas9 system, which belongs to Class 2 (type II), involves the association of crRNA with a single unit of Cas protein (Cas9) for its function [26]. This system, which can potentially edit any gene or genomic region, is widely used, including in virology. The functional complex comprises Cas9 and a single-guide RNA (sgRNA); TracrRNA and crRNA can be fused into a sgRNA. According to recent studies, class 2 type VI CRISPR effector Cas13 can efficiently target and cleave RNA instead of DNA in different model systems, including mammalian cells [27, 28]. Thus, the CRISPR-Cas13 system offers the potential to detect RNA viruses and treat RNA virus infections. Cas13 proteins have been classified into several types, each containing two higher eukaryotes and prokaryotes’ nucleotide (HEPN)-binding domains necessary for RNA degradation [29‐31]. Additionally, Cas13 does not require a protospacer adjacent motif (PAM) sequence, increasing the flexibility of crRNA target sites [32].

While CRISPR/CAS system promises great advances in the genomic and proteomic modification, it still has its own limitations. In spite of being highly specific in-silico, CRISPR/Cas system can show multiple off-target properties, potentially compromising the expected results, especially in-vivo [33, 34]. Furthermore, there have been reports indicating that human cases can develop anti-Cas antibodies, which in turn can limit the use of the CRISPR/Cas pathway in the antibody-positive cases [35].

To identify host factors promoting virus replication, we can perform the loss-of-function (LOF) approach in the permissive host cells and the gain-of-function approach in the non-permissive host cells. By contrast, gain-of-function and loss-of-function strategies are also used in permissive and non-permissive host cells to determine suppressor factors, respectively [36]. We have different CRISPR screens depending on their mechanism of action: CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), and CRISPR knockout (CRISPR-ko) screens. CRISPRi and CRISPR-ko are loss-of-function approaches offering unprecedented opportunities to discover genes (Fig. 1). Before CRISPR was established, insertional mutagenesis in haploid stem cells and RNAi were used to edit genes, providing significant insights into understanding the host factors critical to viral replication and inhibition; nevertheless, the implementation of these techniques is restricted by having serious off-target effects [37]. Furthermore, the loss of function screens cannot reveal all the relevant pathway factors; each screen is conducted in a particular cell line, which may lack the expression or redundancy of certain relevant elements. Gain-of-function screens can overcome these limitations. CRISPRa screening is a gain-of-function approach which uses modified Cas9 protein as a transcription factor [38]. H840A and D10A mutations at HNH and RuvC endonuclease domains create a nuclease, ‘dead’ Cas9 (dCas9) molecule, revoking DNA cleavage, but is still capable of interacting with sgRNA and can bind to targeted DNA sites [39]. dCas9 can fuse to gene activation domains (VPR [40], SunTag [41, 42], SAM [43]), activating gene expression. Notably, CRISPRa is a convenient alternative which allows host-virus geneticists to conduct thorough, potent, and accurate gain-of-function studies compared to cDNA overexpression systems that usually overlook functional gene isoforms [43].

Even though the CRISPR/Cas system can be a very useful method in tracking cellular pathways through loss-of-function and gain-of function studies, the application of its results in the complex organisms and especially in the human body should be done with great caution. Gain-of-function approaches might lead to unwanted activation of potentially harmful pathways, leading to toxic end-products; while loss-of-function methods, through off-target functions, might inhibit vital cellular pathways, leading to cellular damage [33]. These events also happen in-silico and in-vitro, which might lead to a misunderstanding of the studied pathways. Furthermore, any “hit” by the CRISPR screens is considered to be valid, and concepts such as false-negative or false-positive have no place in the interpretation of the screens; this, although makes the CRISPR system specific, it can also make the results difficult to analyze. Thus, this system should be designed as specific to the desired target as possible to avoid any kind of the aforementioned side effects. It should be noted that any identified gene roles in CRISPR studies need to be further confirmed by other experiments outside the context of CRISPR screens; this necessitates follow-up experiments to be carried out via multiple other methods.

Since human pathogenic viruses exploit host factors, investigations are currently exploring the host functions leading to the viral infection. The CRISPR genetic screening strategy has successfully recognized host factors essential for entry, replication, and the spread of viruses [15]. After the Flaviviruses bind to the receptors of the host cell [44] and subsequent clathrin-mediated endocytosis occurs, the viral genome is finally released to the cytosol [45‐47]. The viral RNA by its 5′-cap structure binds to ribosomes, and a viral polyprotein is produced in the translation process anchoring to the endoplasmic reticulum (ER) membrane. The viral polyprotein cleavage by the viral and cellular proteases results in the structural and non-structural proteins of the virus [44, 48]. Remarkably, other viral life cycle processes, such as the viral RNA replication and virion assembly, occur at the ER site. However, despite the awareness about these processes, there is little in-depth knowledge of the involved host proteins. According to the investigations performed so far, CRISPR screens indicated that flaviviruses require ER protein complexes for their replication cycle. These screens have recognized some of the ER proteins needed for the viral replication cycle. Besides, host factors with strong activity against various viruses can also be recognized through CRISPR-mediated screenings. In this regard, many libraries exist for studying particular sets of genes, such as pooled sgRNA libraries for investigating interferon-stimulated genes (ISGs) [49]. In the following sections, we have discussed these factors thoroughly. Host factors involved in viral replication, as well as novel antiviral genes identified using the CRISPR screens, are listed in Tables 1 and 2, respectively.

CRISPR Screens have displayed different cell surface molecules employed by several flaviviruses to enter the cell. Researchers have used CRISPR/Cas screens to investigate the hepatitis C virus (HCV) notably; prominent genes identified in the HCV screens were distinct from those found in other Flaviviridae members. They included genes related to viral receptors, RNA-binding proteins, and enzymes involved in metabolism [14]. It is known that various host factors such as CD81, OCLN, and CLDN1 mediate the entry of HCV into the host cells, which have been identified as critical genes through CRISPR/Cas screens [59, 60]. Based on a CRISPR/Cas9 genome-wide screen, integrin αvβ5 was found to be a ZIKV internalization factor. Significantly, the results showed that αvβ5 blocking antibody or two inhibitors, such as cilengitide and SB273005, can reduce ZIKV infection, indicating that αvβ5 integrin can act as a potential therapeutic target [63]. In another study, two important proviral factors, Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1), through using CRISPRa screen were discovered. It has been demonstrated previously that WWTR1 plays a crucial role in the replication of ZIKV [64]. This study focused on the role of the RhoV factor and found that it plays a crucial role in the viral infection process of several flaviviruses, particularly the ZIKV. It seems that RhoV probably acts through its GTPase activity and its downstream effector protein, Pak1, at the step of endosomal entry, and thus increases the viral infectivity. This finding is considered to be significant since RhoV acts in the early stages of the viral cycle and can be a promising target for antiviral therapy [62].

The signal peptidase complex (SPC) is a membrane complex in the endoplasmic reticulum, where it cleaves signal peptides (SPs) from the N-termini of the secretory and membrane proteins. A subset of signal peptidase complex (SPCS) proteins is required for efficient cleavage of flavivirus structural proteins (prM and E) and the secretion of viral particles; based on the CRISPR/Cas9 screen, Knockout of this factor indicated intense defects in the polyprotein cleavage of several Flaviviridae family members [58]. Another known factor is the translocon-associated protein (TRAP) complex (containing subunits SSR1, SSR2, and SSR3), which facilitates the translocation of proteins across the endoplasmic reticulum membrane [73]. The TRAP complex plays a crucial role in DENV-2, ZIKV, and YFV RNA replication [14]. A recent study has shown that host factors SBDS and SPATA5, which are involved in the formation of ribosomes, are required for the synthesis of viral proteins. Losing these 60S ribosome biogenesis proteins leads to a decrease in the viral replication of flaviviruses and several other viral families. The study specifically found that the loss of function of these two proteins, which are essential for the formation of the 60S ribosome part, causes defects in the processing of rRNAs and the assembly of ribosomes [74].

CRISPR/Cas technology is a growing field in the prevention and treatment of viral infections. The outbreaks of Flaviviridae members in different parts of the world in recent years emphasize the need for innovative methods of vector control which is a strategy used to limit the transmission rate of these viruses. Furthermore, CRISPR/Cas tools are being used to generate gene drives that can potentially decrease mosquito populations. The use of gene drives for mosquito control has attracted much attention in the recent years, as the ease of production of CRISPR-based gene drive systems sets them apart from other methods [88, 89]. There have been multiple methods introduced in CRISPR-based gene drive systems, which include: suppression drives, through which a weakening gene is inflicted in a target population, limiting their activity or even eradicating the targets; and Modification drives, by which the target population is altered in a desired way (e.g. in the setting of malaria control the mosquitoes are altered not to be capable of transmitting the disease) [90].

Recently, CRISPR/Cas9 has revolutionized the study of host-virus biology. CRISPR/Cas technology is utilized to improve our knowledge of how viruses exploit their hosts, as well as to develop new antiviral therapies. Particularly, further studies are constantly conducted using this approach to improve our understanding of flavivirus life cycles, leading to the discovery of essential factors. However, there are still numerous obstacles that need to be addressed. Viral host factor requirements may differ based on viral strains and cell types. In this regard, studies investigating host-virus interactions using CRISPR must use clinically and epidemiologically important virus isolates and corroborate the results with numerous strains. It is also possible that host factors vary from one cell type to another; for example, ZIKV, which infects specialized cells like neural stem cells. Therefore, CRISPR screens should be conducted in the proper cell contexts to reveal the involved factors comprehensively [104]]. Undoubtedly, future screens will provide insight into how viruses have evolved to exploit and subvert host functions. In addition, the findings may lead to potential targets for antiviral therapy. Finally, we expect that next generation CRISPR approaches alongside other new technologies will help us better understand complex biological processes.