Potential of CRISPR/Cas system as emerging tools in the detection of viral hepatitis infection

HAV was first identified in 1973. It is a non-enveloped, icosahedral, single-stranded RNA virus belonging to the picornavirus family. At present, HAV can be divided into three genotypes including I, II, III, with two subtypes including subtypes A and B, which can infect humans. It is usually transmitted through the fecal-oral route, either by person-to-person contact and eating or drinking contaminated food or water. HAV is endemic in many countries, especially countries with limited health care resources [21]. The prevalence of HAV infection is low in developed countries. The seroprevalence of anti-HAV antibodies in the United States is approximately 10% in children and 37% in adults. More than 50% of cases in developed countries are the result of infection from travel to endemic countries [22]. HAV can cause an acute infection that is usually self-limiting and does not lead to chronic infection or chronic liver disease. The incubation period for the HAV is between 15 and 50 days (28 days) from exposure. Individuals with underlying chronic diseases are at higher risk of developing acute liver failure due to HAV infection. The case-fatality rates estimate from 0.3 to 0.6% for all ages and it increases to 1.8% in patients over 49 years old [23]. The spread of HAV in the liver occurs through the portal vein (the blood vessel that brings blood to the liver from the intestines) after the virus passes through the mucosa of the small intestine wall. The viruses subsequently replicate in hepatocytes before excretion, again via bile through feces [24]. Acute hepatitis A is mainly diagnosed by the presence of immunoglobulin M antibody to HAV (IgM anti-HAV). Anti-HAV IgM becomes detectable a few days before or concurrently with the onset of symptoms. Serum IgM levels increase during acute infection and remain positive for an average of 4 months after the onset of symptoms. IgG anti-HAV appears early in the course of infection and is detectable for many years, conferring lifelong immunity. Although Nucleic acid amplification techniques (NAAT; such as PCR) are more sensitive than the immunoassay test for the detection of HAV, they are rarely used for this purpose. Sequencing and phylogenetic analysis are mainly used to trace outbreaks, and these techniques are particularly useful for identifying transmission routes [24, 25]. Currently available vaccines for HAV are safe and highly effective and are included for routine childhood immunization programs in different countries [26]. The standard vaccination schedule for the inactivated vaccine against HAV consists of two doses administered at least 6 months apart. This vaccination schedule has been shown to be highly protective and is expected to last for many years [27].

HBV is a prototype member of the Hepadnaviridae family. It is an enveloped viruses which contains relaxed, circular, partial double stranded DNA [28]. Although HBV is a DNA virus, replication occurs through reverse transcription from pre-genomic RNA (pgRNA) [29]. Currently, HBV is classified into 10 genotypes, including genotypes A to J, based on differences in the genome sequence, with 35 sub-genotypes. The distribution of genotypes varies widely around the world [30]. HBV is one of the common causes of liver diseases including liver cancer and is highly contagious; it is transmitted through exposure to infected blood and other body fluids (particularly amniotic fluid, semen, and vaginal fluids) [31, 32]. It is 100 times more infectious than HIV and 10 times more than HCV, as well [22]. The prevalence of HBV infection varies in different regions of the world, ranging from 0.7% of the adult population in low-endemic areas such as North America, Western Europe, and parts of South America to 6.2% in high-endemic areas such as Africa, Southeast Asia, and China [30]. The overall prevalence of chronic HBV in the USA was about 3/5% [33]. HBV infection can lead to both acute (short-term) and chronic (long-term) disease. Acute HBV infection can be asymptomatic or symptomatic. Most infected adults recover, even if their signs and symptoms are severe, but 5–10% of infected adults develop chronic infection, potentially leading to cirrhosis and liver cancer [34]. HBV has a specific tropism for liver cells to which it adheres and fuses during primary infection. HBV is internalized into hepatocytes by endocytosis after binding to its high-affinity receptor, sodium taurocholate co-transporting polypeptide (NTCP). The capsid of the virus then released into the cytoplasm, and translocated to the nuclear pore via the microtubule network. At the nuclear pore, rcDNA is released in the nucleus where it is converted into covalently closed circular DNA (cccDNA). In this step cccDNA becomes chromatinized, and serves as the transcriptional template for all HBV pregenomic RNAs. In the cytoplasm, pgRNA is encapsidated and retrotranscribed into rcDNA. The capsids are either enveloped and secreted as new virions from the cell, or transported back to the nucleus to amplify the pool of cccDNA [35]. HBV DNA encoded several different proteins play an essential role in viral persistence and liver pathogenesis, including two core proteins, a particulate core antigen (HBcAg), and a secreted antigen (HBeAg), surface antigen (HBsAg), HBx and polymerase and all of which play a role in its diagnosis and surveillance [24, 36]. After infection, HBV DNA and HBsAg can be detectable in an infected person’s serum within 1 to 2 weeks. Six to eight weeks later, IgM anti-HBc and HBeAg become detectable. In acute HBV infection cases, HBsAg and HBeAg disappear within 4–6 months. The disappearance of HBsAg is followed by the appearance of Antibody to HBsAg (Anti-HBs). The interval between the disappearance of HBsAg from the serum and the appearance of anti-HBs in it is known as the window phase and may last up to 6 months [22]. Hence, the initial step of HBV diagnosis is performed using a serological test to detect HBsAg and other HBV antigens and antibodies. Further molecular tests for quantitative and qualitative detection are used to verify the first step of diagnosis (Table 1) [37]. Reactivation of HBV refers to a sudden increase in HBV replication in a patient with chronic or previous HBV. Although, HBV reactivation (HBVr) can occur spontaneously, it is more commonly induced by the immunosuppressive drug therapy (ISDT) for cancer, immunologic diseases, or transplantation [38]. Vaccination is a highly effective strategy for HBV infection prevention. A three-dose series of vaccination of Hepatitis B vaccines (Recombinant), composed of HBsAg, results in a protective level of anti-HBs titers in > 90% of healthy individuals [39].

HCV, a member of the Flaviviridae family, has a positive-sense single-stranded RNA genome coding three structural and seven non-structural proteins. The high mutation rate of HCV leads to marked genomic heterogeneity. HCV is classified into seven confirmed genotypes and at least 67 confirmed subtypes. Meanwhile, genotypes 1–3 are globally distributed, while genotypes 4–6 are concentrated in a certain area. HCV genotype 4 and subtype 5a are endemic in the Middle East and northern parts of South Africa, respectively. HCV genotypes 6 and 7 are mainly observed in Southeast Asia and the Democratic Republic of Congo, respectively [24, 40]. The main forms of HCV transmission are through the blood borne route, such as intravenous and intranasal drug use, contamination of medicine injections, and by sexual contact [41]. The worldwide prevalence of HCV is estimated to be 1.5–2.3% in the most affected areas and 0.5–1.0% in other areas [42]. The prevalence of HCV in injection drug users is between 50 and 90%, which is the largest group among infected people [43]. HCV can cause acute infections with a high tendency for chronic disease. Chronic HCV can lead to severe liver disease including cirrhosis and the risk of HCC [44]. Scavenger receptor class B type I (SCARB1), Occluding (OCLN), Claudin-I (CLDN1) and Cluster of differentiation 81 (CD81) are the main host factors that facilitate the attachment and uptake of HCV into hepatocytes. Furthermore, CD81 interacts with the viral particle through the viral envelope glycoprotein E2 or other molecules, facilitating the entry of the viral particle into the hepatocyte [45]. Virological diagnosis of HCV is established by two categories of laboratory tests, including indirect tests, in which serological assays are performed to detect a specific antibody of HCV (anti-HCV) in the infected patient, and direct tests, in which the detection, quantifying, or characterization of the components of HCV viral particles such as HCV RNA and core antigen can be conducted. The sensitivity and specificity of third generation enzyme immunoassays (EIAs) for detecting antibodies against different antigens of the HCV viral particle is 99% after 2 to 6 months following exposure to the virus. One of the direct assay methods is the use of real-time PCR for the quantitative or qualitative detection of HCV RNA, which can detect HCV particles in patients with at last 10–15 IU/mL (Table 2) [46].

HDV, a member of the genus Deltavirus, is an enveloped, circular, single-stranded negative-sense RNA virus that encodes two kinds of proteins; the small protein (S-HDAg) and the large protein (L-HDAg), which are 24 kDa and 27 kDa in size, respectively. S-HDAg is required for viral RNA replication, while the L-HDAg is essential for viral assembly [47]. HDV is considered a satellite virus because it requires the assistance of HBV surface proteins (HBsAg) to generate mature virion particles. As a result, two distinct patterns of infection for HDV can establish; either HBV/HDV coinfection or HDV superinfection. HBV/HDV coinfection occurs when a person simultaneously becomes infected with both HBV and HDV, whereas HDV superinfection occurs when a person who is already chronically infected with HBV acquires HDV. Development of a chronic HDV superinfection can exacerbate hepatic injury caused by HBV, and both coinfection and superinfection have been shown to result in more severe outcomes than HBV infection alone, including fulminant hepatitis, HCC, and chronic hepatitis [48]. Currently, three genotypes of HDV have been identified (Genotype I–III), among them Genotype I is predominant [22]. Like HBV and HCV, HDV is primarily transmitted sexually, as well as through exposure to infected blood and blood products [28]. Globally, it is estimated that about 5–10% of patients with chronic hepatitis B (CHB) are co-infected with HDV, with a higher prevalence in injecting drug populations. Areas with a high prevalence of HDV infection include the Mediterranean basin, South America, Mongolia, and Sudan. In North America and Northern Europe, the prevalence is low and mostly confined to injecting drug users [22, 49]. The first step in the diagnosis of HDV infection is testing HBsAg-positive individuals for the HDV-specific total antibodies (combined IgG and IgM) in the serum. For anti-HDV- antibodies positive patients the next step is testing for HDV RNA in serum to determine whether the antibody represents an ongoing active HDV infection (HDV-RNA-positive) or only represents a serologic scar (HDV-RNA-negative) [50].

HEV was first discovered in 1983 when scientists were looking for the cause of the outbreak of non-A, non-B (NANB) hepatitis, which is transmitted enterically [51]. HEV belongs to the Hepevirus genus of the Hepeviridae family. [52, 53]. Three groups of mammalians Hepeviridae have been recognized based on full genome sequencing from human and animal strains. The first category consists of viruses that affect people, pigs, deer, and rabbits. The 24 sub-genotypes of the four human HEV genotypes (genotypes 1–4) identified from infected patients are included in this group [53]. HEV is most often transmitted through contamination of water and food, and less commonly through the zoonotic route or through blood transfusion [52]. HEV genotypes 1–4 show a selected geographical distribution. Genotypes 1 and 2 are the main causes of acute HEV infections in endemic areas in South Asia and Sub-Saharan Africa [54]. Genotype 3 is the most common genotype associated with HEV infection in Europe and America [55]. HEV genotype 4 was initially identified only in Asia, but further reports have also identified this genotype in pigs and humans in Europe [52]. HEV genotypes 1 and 2 are obligate human pathogens and are mainly transmitted through contaminated drinking water in areas with poor sanitation, while genotypes 3 and 4 are zoonotic and mainly transmitted through the consumption of raw or undercooked meat or liver products from its main host (i.e., pig, wild boars and deer) [56]. HEV infection is generally self-limited, and causes acute hepatitis. Infection with HEV has an incubation period of 15 to 60 days. Typical acute HEV may lead to elevation in aminotransferases to greater than 10 times than normal. The main clinical symptoms described are anorexia, nausea, malaise, and abdominal pain, in addition to arthralgias, diarrhea, pruritus, and a rash. Within 1 to 6 weeks after the infections, aminotransferases return to normal. The case fatality rate is approximately 0.5 to 3% in adult individuals. HEV can also cause fulminant hepatic failure (FHF), in which encephalopathy within days or weeks of the onset of symptoms develop. FHF appears to occur more commonly in pregnant women, hence HEV has a higher case fatality in pregnant women [22]. Both serological tests and nucleic acid-based tests have been developed for the diagnosis of HEV for epidemiological and diagnostic purposes. Serological assay for HEV infection usually depends on the detection of anti-HEV IgG and anti-HEV IgM. Anti-HEV IgM antibodies are usually detectable in the acute phase of the disease and its presence in serum lasts for approximately 4 or 5 months, indicating recent exposure, while anti-HEV IgG antibodies can persist for more than 10 years, indicating prior exposure. Therefore, the diagnosis of acute HEV infection is based on the presence of anti-HEV IgM, while the anti-HEV IgG diagnostic test is a good option for epidemiological investigations [57]. However, detection and quantification of HEV-RNA by PCR is a gold standard approach for the diagnosis of acute and chronic HEV infection [58]. Currently, no specific vaccines, except the one licensed in China, or therapeutic options are available for HEV [22].

Since the initial discovery of the CRISPR–Cas systems, its various variants have expanded rapidly. Currently, CRISPR-Cas systems are divided into two classes, six types and several subtypes based on evolutionary relationships. Depending upon the nature of the effector ribonucleoprotein complexes, two main classes of CRISPR-Cas systems have been defined; Class 1 (includes Types I, III, and IV) and Class 2 (includes types II, V, and VI). Class 1 systems are characterized by a complex of multiple effector proteins, and class 2 systems encompass a single crRNA-binding protein [11]. Most of the identified CRISPR-Cas systems (about 90%) belong to class 1 systems [16]. Among the diverse CRISPR systems, class 2 systems have primarily been applied for diagnostics, as these systems are simpler to reconstitute. They include enzymes with collateral activity, which serves as the backbone of many CRISPR-based diagnostic assays. Cas9, Cas12 and Cas13 have a major impact on the CRISPR-Cas classification for type II, type V and type VI as unique signature effector nucleases, respectively. The latest classification of CRISPR-Cas systems attributes the largest number of subgroups to the type V system with 17 derivative subgroups [11]. Class 1 systems (such as the type III effector nuclease Csm6 or Cas10) have also been engineered for diagnostics, either in combination with components of the class 2 system or with the native type III complex) [66]. A RuvC-like nuclease domain in the Cas12 system catalyzes the cleavage of a dsDNA target by Type V systems [67]. This feature has led to the use of Cas12a, also known as Cpf1, in viral pathogen diagnosis tests [68, 69]. The CRISPR-Cas type VI system has five subtype variants. Cas13a in subtype VI-A is known as an RNA-guided Rnase [16]. Cas13a is able to bind to crRNA and thus form a complex that eventually cleaves ssRNA [12]. The ability of Cas13a in ssRNA cleaves has led to the development of modern platforms for the rapid and sensitive detection of infectious diseases. Each of the Cas12a and Cas13a-based viral infection diagnosis platforms is discussed in the next section (Fig. 1) [70, 71].

Owing to the programmable nature of CRISPR-Cas systems, they have been exploited in many different fields of biology and medicine and, in fact, have revolutionized these fields. An increasing number of studies have shown that CRISPR/Cas technology can be integrated with biosensors and bioassays for molecular diagnostics. CRISPR-based diagnostics has attracted much attention as highly specific and sensitive sensors with easily programmable and device-independent capabilities. This technique has been used for the sensing of nucleic-acid-based biomarkers of infectious and non-infectious diseases and for the detection of mutations and deletions indicative of genetic diseases. With further research, it holds promise for detecting other biomarkers such as small molecules and proteins [66, 72]. In this section we briefly describe the application of CRISPR/Cas systems in the field of diagnostics.

Accurate, sensitive, and rapid diagnosis of viral infections is the critical first step toward accurate and timely treatment of viral infections. CRISPR-Cas-based diagnosis of pathogens has gained tremendous popularity over the past few years, mainly due to the high specificity, sensitivity, and rapid diagnosis of this system [81]. Numerous variations and advancements have been introduced to the CRISPR-based platform, counting on the collateral activity of CRISPR- Cas type V and type VI proteins, but the general conception remains unchanged. In the following, we outlined the various platforms based on the CRISPR-associated nucleases Cas9, Cas12, or Cas13 using for diagnosis of different viral infections.

Chen X et al. devised a CRISPR-Cas based detection platform, known as “CRISPRHBV”, for ultrasensitive and early detection of two major HBV genotypes (HBV-B and HBV-C) in clinical applications. CRISPR-HBV consists of three steps including multiple crossover displacement amplification (MCDA) for rapid pre-amplification, Cas12b-based detection to decode targets, and result readout with real-time fluorescence and lateral flow biosensors. This system is able to detect 10 copies of genomic DNA per test and has 100% specificity and also has no cross-reactivity in other HBV genotypes and pathogens. The entire assay process, which includes DNA template extraction, MCDA preamplification reaction, CRISPR-Cas12b-based detection, and readout of results can be completed in 60 min and does not require expensive equipment. Additionally, the feasibility of the CRISPR-HBV assay for HBV-B and -C genotyping was successfully confirmed by validation with clinical samples. Therefore, the CRISPR-HBV system has the potential to be a POC diagnostic test for the detection and diagnosis of HBV genotypes B and C in clinical applications, especially in countries with insufficient medical resources [92].

Since the formation of an intranuclear reservoir of cccDNA in the hepatocyte is the main cause of persistent HBV infection, Zhang, X et al. developed highly sensitive and specific diagnostic tools for the detection of HBV cccDNA based on CRISPR-Cas13a technology. This technique includes the following steps: (1) using plasmid-safe ATP-dependent DNase (PSAD) and HindIII enzymes to digest circular rcDNA and linear double-stranded DNA, (2) amplification of specific HBV cccDNA fragments using rolling circle amplification (RCA) and PCR, and (3) target detection using the CRISPR-Cas13a system. This assay can detect 1 copy/µL HBV cccDNA by CRISPR/Cas13-assisted fluorescence readout. Furthermore, by performing ddPCR, qPCR, RCA-qPCR, PCR-CRISPR and RCA-PCR-CRISPR, this assay was validated for the detection of cccDNA in HBV-associated liver tissues, plasma, whole blood, and peripheral blood mononuclear cells (PBMCs) [93].

Many studies revealed that drug-resistance mutations occurring in the HBV genome can increase the risk of HBV transmission or cause active replication of the viral genome, resulting in a lytic infection and other clinical problems in affected patients. One of the most important factors in antiviral drug resistance is the mutation in the YMDD motif of the HBV P gene. Therefore, Wang S. et al. developed a highly sensitive and practical method for the detection of HBV DNA and YMDD drug resistance mutation based on the CRISPR-Cas13a detection system and PCR amplification, and evaluated its diagnostic capability using clinical samples. This technique represents very high sensitivity for detecting HBV and identifying drug resistance mutations using conventional PCR. Given that PCR amplification is more stable than isothermal amplification techniques, PCR was used to target amplification prior to CRISPR-Cas13a detection. In terms of sensitivity, it was found that this technique can detect one copy per test for HBV DNA and YMDD drug resistance mutations [94]. In another study, Ding, R et al. developed a fast and sensitive HBV POC test based on LAMP-Coupled CRISPR-Cas12a. This assay creatively solves the problem of the POC technique within 10 min, especially the nucleic acid sample extraction problem. Based on Loop isothermal amplification (LAMP)-Cas12a, visualization of assay results is provided with both fluorescent and lateral flow test strips. High-sensitivity real-time detection can be achieved in fluorescence readout, while results visible to the naked eye can be achieved with lateral flow test strip technology. The fluorescent readout-based Cas12a assay can achieve HBV detection with a sensitivity of 1 copy/µL in 13 min, while the lateral flow test strip technique takes only 20 min. The evaluation of clinical samples shows the sensitivity and specificity of both the fluorescence readout method and the lateral flow test strip 100%. Additionally, the assay results were completely comparable with qPCR. The LAMP-Cas12a-based HBV technique relies on minimal equipment and low costs to provide rapid and accurate test results, thereby offering significant practical potential for POC HBV detection in medically underserved regions (Table 3) [95].

The genetic diversity of HCV and quasispecies generated during replication have led to viral resistance to direct-acting antivirals (DAAs), as well as obstacles to the development of a vaccine. The accurate and timely diagnosis of disease is a prerequisite for efficient therapeutic intervention and epidemiological surveillance [96]. While techniques such as immunoassay and qPCR play an important role in the diagnosis of HCV, rapid and accurate POC testing is important to identify individuals with HCV, particularly in resource-limited settings where access or availability of molecular testing is still limited. Therefore, to fill this gap, there is a need to develop a new molecular assay for the rapid detection of HCV RNA in resource-limited settings [97]. Recently, Kham-Kjing et al. developed and evaluated a fast and sensitive method for the detection of HCV RNA. The method was based on a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-coupled CRISPR–Cas12 system that allows the detection of specific HCV genome sequences. Amplified products after cleavage reactions can be visualized on lateral flow bands or measured with a fluorescence detector. When tested on clinical samples from individuals infected with HCV, HIV, or HBV, or from healthy donors, the CRISPR-Cas12 assay combined with RT-LAMP compared to the reference method, which was Roche COBAS AmpliPrep/COBAS TaqMan HCV Test, showed 96% sensitivity, 100% specificity and 97% agreement. This assay was able to detect HCV RNA concentrations as low as 10 ng/µL. Therefore, this accurate and specific technique has the potential to be a cost-effective and reliable POC test to identify individuals with HCV in low-resource settings (Table 3) [98].

These overall studies proved the potential of the CRISPR/Cas system as an effective diagnostic tool for viral hepatitis infections. Beyond these studies describe above, till now, no CRISPR-based diagnosis method has been developed for the detection of HAV, HDV, and HEV. However, Cas12a enzyme is the enzyme of choice for developing a CRISPR-based method for HAV detection. As well as, since HDV is an RNA-virus, Cas13a is the choice enzyme for CRISPR-based diagnosis method for HDV. Like HDV, HEV is an RNA-virus, so Cas13a is the choice enzyme for CRISPR-based diagnosis method for the detection of this virus. Uncertainty, the potential of the CRISPR/Cas system can soon become the leading diagnostic tool for different viral hepatitis infection.