To amplify and recognize a viral sequence, DNA- and RNA-based diagnostics currently use PCR or isothermal amplification. Nucleic acid-based methods such as quantitative or qualitative PCR (qPCR) or reverse transcriptase qPCR (RT-qPCR) are the gold-standard methods because the design of qPCR assays is simple but requires knowledge of the viral sequence. However, the cost, sample-to-answer time, and personnel and equipment requirements limit widespread deployment. Numerous isothermal amplification methods for virus detection have been developed and implemented in order to eliminate the need for costly thermal cycling equipment. These strategies include nicking enzyme amplification reaction (NEAR) [67], recombinase polymerase amplification (RPA) [68], nucleic acid sequence-based amplification (NASBA) [69], loop-mediated amplification (LAMP) [70], and nucleic acid sequence-based amplification (NASBA). Each strategy involves trade-offs with regard to performance traits such as multiplexibility, readout accessibility, sensitivity, specificity, and testing throughput [70,71,72].

Because CRISPR-based diagnostic tools are very precise and sensitive but do not need expensive laboratory equipment, they can enhance conventional procedures. Because Cas–crRNA complexes are inherently sequence-specific, CRISPR-based technologies can be as specific as PCR. In CRISPR/Cas-based diagnostics, Cas12 and Cas13′s distinctive properties are crucial. The collateral cleavage of Cas13 and Cas12 does not demand temperature cycling [73,74]. Additionally, visual readout-compatible reporters can be used to detect collateral cleavage, negating the requirement for pricey apparatuses such as thermocyclers and fluorescent readers [75]. The adaptability of CRISPR-based detection technologies for detecting viral nucleic acids is highlighted by the fact that they have been built for a broad range of both DNA and RNA viruses and make use of various sample processing and amplification techniques, Cas effector proteins, and readouts.

Innovative techniques were created that utilised Cas9′s selectivity for identifying viral genes or differentiating between viral strains before the discovery of Cas effector proteins with collateral cleavage activity. Cas9 cleavage was linked with PCR in a technique known as CARP (Cas9/sgRNA-associated reverse PCR), also referred to as ctPCR, to identify specific viral targets (CRISPR-typing PCR). Using PCR, a specific target sequence including two physically separated Cas9 PAM sites was amplified. Two Cas9 sgRNAs may then be used to target the dsDNA that the initial PCR produced in adequate quantities. Versions 1.0, 2.0, and 3.0 of the ctPCR iteratively improved the reactions needed to detect the presence or absence of the target after Cas9 cleavage.

Through PCR amplification of ligated adapters [76], PCR amplification using reverse primers that only amplified a cleaved and ligated region [77], or qPCR amplification in which the relative efficacy of reactions with and without Cas9 was examined, targets were identified [78]. In addition to ctPCR, NASBA-CRISPR cleavage (NASBACC) was created, which combines NASBA amplification, toe-hold sensors, and Cas9 cleavage. It was necessary to have at least one divergent site that interferes with Cas9′s PAM between the sequences to be distinguished in order to execute NASBACC. If Cas9′s PAM was present in the target, it was able to cleave the toehold sensor binding site, allowing for differentiating signals between the two targets [79].

A few viral strains have been subjected to these Cas9 detection techniques. ctPCR is used to identify human papillomavirus (HPV) genes in various subtypes of HPV16 and HPV18. Additionally, the Zika virus strains from Asia and America were distinguished using NASBACC (ZIKV). These investigations show how versatile Cas9 is for identifying and classifying viruses, but their applicability is constrained by the number of reactions required for either amplification or manipulation of the amplified products.

As a result of the characterization of Cas13 and Cas12′s collateral cleavage activity, a number of user-friendly, CRISPR-based detection techniques with the potential for field deployment and massive scaling have been developed [80].

The technology named SHERLOCK (selective high-sensitivity enzymatic reporter unlocking) was created when the collateral activity of Cas13 was identified [81,82]. Leptotrichia wadei (LwaCas13a) was used in SHERLOCKv1 because it had the best target-activated collateral cleavage activity at the time and was programmable, RNA-guided, and active. The isothermal amplification technique RPA was used to boost the assay’s sensitivity because LwaCas13a alone was unable to identify the vast range of probable virus titres in patient samples [68]. Sherlock needed T7-mediated in vitro transcription of the amplified product because RPA yields a dsDNA product, which prevents Cas13 from detecting it. By introducing a synthetic ssRNA molecule flanked by a quencher and an attached fluorescent dye, the amplified target was detected using the fluorescence signal produced by the associated fluorescent dye.

Soon after, other systems such as DETECTR (DNA endonuclease targeted CRISPR trans reporter) [73] and HOLMES (1-h low-cost multifunctional highly efficient system) began to utilise Cas12 for nucleic acid detection [83]. Because Cas12 can detect the immediate result of amplification reactions, these techniques did not require in vitro transcription following amplification (i.e., dsDNA). An inserted ssDNA-quenched fluorescent reporter molecule was cleaved in trans when Cas12′s collateral cleavage activity was triggered by the detected dsDNA target. Different amplification techniques were used by these two Cas12-based technologies; in their first publications, DETECTR used RPA while HOLMES used PCR. These CRISPR-based detection techniques can be used in a variety of settings, including cancer and human genotyping [84].

Singleplex tests have demonstrated the ability of Cas13- and Cas12-based diagnostics to sensitively identify a variety of viral targets. SHERLOCK’s capacity to recognise small amounts of ZIKV in artificial lentivirus samples at known concentrations and in patient samples with a variety of viral titres was first proven during the Zika epidemic [81].

In a collection of 25 patient samples, DETECTR was initially utilised to identify the DNA of HPV16 and HPV18. With the exception of two samples, DETECTR produced concordant results when compared to the gold-standard qPCR [73]. Since then, a growing number of CRISPR-based assays have been created and approved for use with human viruses, including the ones that cause Lassa fever, the Epstein–Barr virus, the Powassan virus, the H7N9 influenza virus, the hantavirus, the Ebola virus, and the Japanese encephalitis virus (JEV) [85,86,87]. CRISPR-based tests could be created for any viral pathogen given enough genomic data due to the adaptability of these platforms [88,89]. Once a new virus’ genomic sequence is known, CRISPR-based detection techniques can be quickly tested and validated for it. The advent of SARS-CoV-2 in late 2019 served as a prime example of this. New assays were being created and posted on social media and preprint servers soon after the first SARS-CoV-2 genomes were released [90,91,92,93], and soon after that, peer-reviewed papers. The DETECTR approach was used to create a SARS-CoV-2 assay, which was then validated on more than 70 patient samples, demonstrating how quickly these assays can be created [90]. Similar to this, a SHERLOCK test with excellent agreement with RT-qPCR was validated on more than 150 patient samples in Thailand [94]. Soon after, many more publications appeared [95,96,97]. The FDA’s emergency use authorisation (EUA) process for CRISPR-based SARS-CoV-2 diagnostics was also facilitated by the COVID-19 pandemic. The first FDA authorizations of a CRISPR-based diagnostic came from Mammoth Biosciences and SHERLOCK Biosciences shortly after these CRISPR-based detection technologies were published, underscoring the future potential of CRISPR-based diagnostics for becoming a standard selection of molecular assays for viral diagnosis [98].

The field of viral infection is the one where CRISPR-based diagnostic methods have received the greatest attention [99]. The CRISPR/Cas12a and Cas13a families have inspired the development of several research techniques called DETECTR and SHERLOCK, respectively [100]. In a three-step procedure, DETECTR employs the Type V Cas12a enzyme to connect directly to DNA targets [101]. Usually, a guide RNA drives the Cas12a enzyme to a highly sensitive and specific genome’s double-stranded DNA sequence [73]. Once coupled to its viral genetic target, the Cas12a enzyme indiscriminately cleaves a single-stranded DNA molecule connected to a quencher molecule and a reporter fluorescence [100]. This “collateral” cleavage is recognized by the release of a fluorescent signal from the fluorophore and quencher [73]. The DETECTR method’s main benefit is its great sensitivity, which allows it to identify a single viral particle molecule inside a microliter of the sample [100]. The Type VI Cas13a enzyme is used in the SHERLOCK method to bind and cleave RNA indiscriminately using targets that are crRNAs. When certain sequences are present, target RNA is bound by a target-specific molecule with an attached fluorophore, which then cleaves it collaterally, producing a fluorescence signal that can be recognized and studied to determine the presence of viral nucleic acid [102]. Since its inception, For use in recognizing and diagnosing viruses, SHERLOCK has undergone significant research [75]. Researchers have further improved the approach, creating a more straightforward and focused SHERLOCKv2 protocol [75]. The additional CRISPR-associated Csm6 enzyme was paired with Cas13 enzymes, which more than tripled sensitivity [75]. In both laboratory and clinical settings, viruses can be identified using the DETECTR and SHERLOCK procedures. Although it can be used to diagnose any virus, the DETECTR technique has been widely used to diagnose HPV [102]. Recombinase polymerase amplification (RPA) can enhance highly contagious component multiplication and detection when combined with the SHERLOCK and DETECTR methods. [102]. Additionally, the “SHERLOCK” methodology can be improved for the analysis of HIV, a viral disease that is still a major problem for the entire world [100]. According to HUDSON protocol researchers, universal-flavivirus RPA and crRNAs unique to a particular viral species can both be used to pinpoint conserved sections in these viruses’ genetic material [103]. Although any virus can be detected using SHERLOCK and HUDSON protocols, earlier research concentrated on the detection of flaviviruses such as Dengue, Zika, West Nile, and yellow fever viruses [88,89]. How CRISPR techniques can be used to diagnose the new coronavirus (SARS-CoV-2), an emerging pathogen that has infected over 12.9 million individuals and killed over 500,000 people thus far [104], is of great acute interest to scientists at the moment [22]. The lengthy incubation period is also concerning, as a person with the virus may go up to two weeks without signs before exposure to the disease [105]. In the applications presented, the DETECTR approach has been employed to detect this virus and emphasises determining the occurrence of the N and E gene variations unique to SARS-CoV-2 [106]. If both genes are found, a positive result is produced, and the process has been refined to eliminate false positives brought on by related coronaviruses [93]. Several kits have been created by the CRISPR-associated nucleases Cas9, Cas12, or Cas13, including CASLFA, FELUDA, DETECTR, HOLMES, SHERLOCK, and others [107].

The Papovaviridae family of tiny, double-stranded DNA viruses has about 150 different varieties that have been found thus far [14]. E1–E8 primary viral regulatory proteins, two late capsid proteins, and nine or ten open reading frames (ORFs) are all encoded by the approximately 8 kbp long HPV genome (L1 and L2). Due to their sexual transmission, epithelial tissue tropism, and carcinogenic potential, HPVs have an essential role in human illnesses and public health [108]. Continuous speculative-type HPV infection, such as HPV-16 and HPV-18 [109], is strongly linked to the occurrence of cervical cancer in females [110]. Due to the virus’s capacity to lower activity in a host cell to evade host immune surveillance and the difficulty of removing a viral genome [111] from an infected host cell in a latency state, there is currently no medicine for HPV infection that can achieve a satisfying outcome [112]. Retinoblastoma protein (pRB) and the cellular tumour suppressor p53 is inhibited by the HPV E6 and E7 genes, respectively [113]. Therefore, through the activation of cellular oncogenes, overexpression of E6 or E7 caused by HPVs has a significant likelihood of resulting in the malignant transformation of human cells (e.g., ras or fos) [114]. There are now three HPV vaccines available. The bi- and quadrivalent vaccinations have provided protection against the two most common HPV oncogenic genotypes since 2006 (types 16 and 18) [115]. The year 2014 saw the approval of a nine-valent vaccine that offers defence against five additional cancer-causing HPV strains in addition to types 16 and 18. The male-approved vaccinations quadri- and nine-valent provide defence against the genital wart-causing HPV strains 6 and 11 [116]. In addition to vaccine cost, a major barrier to the acceptance of the HPV vaccine is the lack of experience providing a two-dose vaccine to girls between the ages of 9 and 14 through routine immunisation programmes [117]. Impact studies have shown a considerable reduction in the prevalence of oncogenic and other genotypes present in the immunisation as well as high-grade precancerous lesions and genital warts. The implementation of an HPV vaccine in low- to middle-income countries has significant financial challenges [118].

Despite significant advancements in various HPV treatments, there is still a pressing need to create new, effective therapeutics for the carcinogenesis caused by HPV [119]. CRISPR/Cas9-based gene therapy for HPV infection is now a reality due to recent technological advancements. To damage the HPV genome, multiple studies have thus far described anti-HPV applications of the CRISPR/Cas9 system [119]. According to the findings, the CRISPR/Cas9 strategy offers a great deal of potential for advancement as a clinically useful treatment for disorders linked to HPV [120]. There are various HPV life cycle editing targets for CRISPR/Cas9. To enhance the therapeutic effects, CRISPR-related technologies still need to be developed [14].

HBV is still a health problem, as seen by the 350–400 million chronic HBV carriers estimated worldwide [14,121]. The family Hepadnaviridae is seen in people with persistent HBV infection. The hepatitis virus is a hepatotropic DNA virus that can lead to liver cancer and cirrhosis. It replicates by reverse transcription in host hepatocytes at the stage of RNA intermediates [108]. Eight genotypes (A–H) of the HBV genome have been determined taxonomically, and between any two of these, there are over 8% nucleotide variations [122]. Given the low likelihood of sustained viral response (SVR) or cure in HBV-infected individuals, novel and more potent HBV treatment regimens must be developed [103]. A novel method for the anticipation and dealing of HBV infectious illnesses may be possible given CRISPR/Cas9 technology’s rapid development [123]. Gene therapies currently offer the great ability for entering clinical submissions after incapacitating several methodological obstacles and have emerged as a promising prospective treatment for HBV infections, particularly in efficiently targeting cccDNA [124]. Two research teams separately reported suppressing HBV infection in preclinical applications using the gene-editing tools ZFNs or TALENs [125]. Lin et al. first looked into the ground-breaking effort to employ the CRISPR/Cas9 system in preventing HBV infection in vitro and in vivo in 2014 [126]. To successfully suppress viral replication and production, some studies have used specially engineered Cas9/sgRNA (or Cas9/multiplex gRNA) combinations to alter just one locus, which is often in the conserved region of the HBV genome For the objective of eradicating HBV genomes, several other research studies associated with the combination of CRISPR/Cas9 and other techniques (such as various chemicals or inhibitory systems) have also been established [127,128,129,130] A Cas9 variation known as dead Cas9 (dCas9) has also been shown to prevent HBV replication without removing the HBV genome [130,131]. A study was recently carried out to investigate NU7026, a powerful NHEJ inhibitor, which blocked the deprivation of cccDNA-mediated cleavages by CRISPR/Cas9.

Antiretroviral therapy has decelerated the spread of HIV and significantly improved the clinical outcomes connected with this viral infection. The transcriptionally silent but replication-competent provirus survives in a long-lived cell reservoir primarily constituted of memory CD4+ T cells, making a complete cure for HIV infection challenging. This reservoir is highly robust and resistant to antiviral medications as well as immune response effects, posing a considerable hurdle to the complete eradication of HIV infection [135]. HIV, a significant global disease that mostly consists of HIV-1 and HIV-2, calls for cutting-edge treatments [136]. New infections occur every day, according to a recent UNAIDS report. HIV-1 differs from HIV-2 in that it is more transmissible and harmful in the human host [137]. The so-called chronic sickness of AIDS eventually develops as a result of significant CD4+ T-cell depletion brought on by active HIV-1 replication in living organisms [138]. These HIV therapies, however, intended to block different viral life cycle stages [139], are nonetheless unable to cure the illness since HIV-1 has been permanently incorporated into the host DNA. In light of these findings, scientists have concentrated on treating AIDS using CRISPR/Cas9-based gene editing techniques to open up a wide range of new opportunities for HIV-1 prevention and treatment [140]. When a patient tests positive for HIV, highly active antiretroviral therapy (HAART) is usually started as soon as possible. It is made up of three or more antiretroviral drugs taken together. HAART is also known as antiretroviral therapy (ART) and combination antiretroviral therapy (CART). A key component of HAART is the simultaneous administration of several drugs that inhibit viral replication through different mechanisms. This prevents the spread of a virus that has developed resistance to one of the drugs through the combined action of the other two drugs. The Infectious Diseases Society of America describes HAART regimen management as a multifaceted procedure that should be carried out by or in consultation with a practitioner with specific expertise [141,142,143,144]. It should be carried out by or in consultation with a doctor with particular experience, according to the definition of the HIV-Medicine Association of the Infectious Diseases Society of America.

Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), which are the most often prescribed medications for ART, are known to cause fatal lactic acidosis and peripheral neuropathy as a result of mitochondrial toxicity. Several NRTIs can also cause anaemia, lipodystrophy, and bone marrow suppression as unwanted side effects [145,146,147,148,149]. Tenofovir is often well tolerated; however, it can damage the kidneys or lower bone mineral density. Patients may consider other medications if they have a history of osteoporosis or renal impairment (eGFR less than 60 mL/min/1.73 m). It will be required to monitor hepatic function clinically and in the lab because the discontinuation of tenofovir formulations could cause an immediate deterioration of HBV. After using abacavir, patients with the HLA-B*5701 mutations are more likely to develop a CD8-mediated hypersensitivity reaction. Didanosine is rarely used since it can cause hepatomegaly and pancreatitis. Numerous research studies using CRISPR/Cas9 technology as a means for treating HIV/AIDS have been created quickly since the first two CRISPR/Cas9-based applications in the prevention of HIV-1 were reported by Cho and Ebina, respectively, in 2013 [12,150]. Targeting viral genomes and host genes has previously been two crucial strategies for battling HIV-1 infection. Despite the development of Cas9/multiplexed-sgRNA technology, there are yet no studies that specifically and jointly target the two coreceptor genes CCR5 and CXCR4 using CRISPR/Cas9 molecular scissors. The largest barrier to effective HIV infection control at the moment is the purging of latent viral reservoirs. Latent viral reservoirs, which mostly attach to dormant memory CD4+ T cells, can persist for up to 60 years, as seen in HIV patients taking ART therapy [151]. Scientifically speaking, stem cell transplantation (SCT) is not a recommended treatment for HIV/AIDS [152]. These two case reports indicate that SCT was first intended to treat cancer, not HIV/AIDS, in the two individuals. The accidental therapies give hope for the future use of customized gene therapy in the treatment of AIDS [153].

HSV-1, frequently known as human herpesvirus-1, is the original participant in the family of human herpesviruses [154]. The HSV-1 genome has double-stranded DNA that is similar to other herpesviruses [155]. The first work utilizing the CRISPR/Cas9 expertise in contrast to HSV-1 prolific infection in cell culture was published by Roehm et al. Three diverse sections of the viral DNA that encode the “HSV-1 ICP0” protein were chosen as the targets for the three guide RNAs. ICP0 is a crucial HSV-1 immediate early (IE) regulatory protein that has a big impact on the expression and replication of the viral genes [154,156]. According to these findings, ICP0 was inactivated by Cas9/gRNA in cells expressing functional gRNA and Cas9, as seen by the cells’ sharp reductions in the ability to maintain ΔICP0 HSV-1 multiplication [154]. It was discovered that ICP0 antiviral action that disrupts PML bodies is interfered with by mutations brought on by Cas9/Grna [155]. Studies using fluorescence microscopy and biochemical methods revealed that HSV-1 infection and replication were suppressed, as well as ICP0 protein production being inhibited. Plaque assays revealed that Cas9 and gRNA-expressing cells had lower virus titres and proliferation [154]. The TC620 cells’ capacity to advance through the cell cycle, undergo apoptosis, or remain viable was unaffected by the appearance of Cas9 and gRNAs [155]. The anti-HSV-1 Cas9/gRNA systems had little off-target effects, according to SURVEYOR assays and PCR sequencing analysis, since no indel mutations were discovered in many representative human genes identified by bioinformatics screening investigations [154]. These findings imply that there is little cytotoxicity or off-target activity in the Cas9/gRNA system. After being transfected with HSV-1-eGFP, which had a GFP expression cassette, Vero cells were first treated with anti-HSV-1 gRNAs. GFP expression was then analysed as a sign of HSV-1 infection and replication [157]. The majority of gRNAs that targeted crucial HSV-1 genes effectively reduced viral replication [157]. Although the nonessential genes are targeted, this can largely be explained by the fact that the Cas9/gRNA system produced double-strand DNA breaks, rendering these genomes inactive for the formation of viral offspring [155]. The ability of nine gRNAs to alter the topmost three anticipated off-target locations in the human genome was examined to evaluate possible off-target editing by the CRISPR/Cas9 system. These 27 human genome loci were amplified by PCR and sequenced using DNA samples taken from gRNA-expressing and control cells. These loci showed no evidence of CRISPR/Cas9-induced editing, indicating that CRISPR/Cas9-mediated genome editing did not take place at undesirable locations [157]. When HSV-1 was reactivated in the cultured cell model harbouring functional gRNAs, replication was suppressed [157]. These findings align with those of a recent study that used the CRISPR/Cas9 systems to combat HSV-1 lytic infection in Vero cells [158].

Numerous techniques, including RT-qPCR, sequencing-based techniques, and immunological procedures, have been used to diagnose SARS-CoV-2. Common methods of detecting SARS-CoV2 (RT-PCR, serology) have been restricted because of low accuracy and sensitivity of sample preparation, reagents, equipment, and various types of clinical specimens; consequently, more research is needed to find low-cost methods with high sensitivity to detect SARS-CoV2 [159]. The most recent CRISPR/Cas technology can therefore be employed to develop diagnostic or therapeutic procedures. The emergence of novel SARS-CoV-2 strains, highly contagious viruses, and asymptomatic individuals enable the disease to spread throughout the world, wreaking havoc on the healthcare system and the global economy [160]. Therefore, it is highly recommended that quick and precise diagnostic techniques be used to identify infected people and confine them in order to stop the deadly virus’s cycle of global spread. The most well-known genome editing technique, the CRISPR/Cas system, ushers in a new era in SARS-CoV-2 detection. CRISPR/Cas-based methods have high specificity and sensitivity without the need for expensive equipment, in contrast to conventional laboratory methods for detecting COVID-19, such as RT-qPCR and next-generation sequencing (NGS), which demand highly skilled technicians and expensive facilities, and serological tests that recognise antibodies specific to SARS-CoV-2 in later stages of infection. CRISPR/Cas-based methods would be ideal for simple tests that are crucial for the diagnosis of COVID-19 because of their high precision, specificity, portability, and minimal equipment requirements, particularly in developing nations or locations with a higher risk of infection, such as airports, ports, and emergency rooms [161,162,163,164,165].

Plants including herbs and crops are susceptible to a number of viral infections that can cause significant economic losses [166,167]. CRISPR/Cas9 technology manipulates plant viral defence mechanisms by recognising and deleting pathogenic genes that infiltrate them. It can also be used to develop agricultural cultivars with increased tolerance to several plant viruses [168]. The use of association genetics in plant breeding, with an emphasis on single nucleotide polymorphisms (SNPs) and other widespread molecular markers, has increased and produced vital high-throughput data for the detection of quantitative trait loci (QTLs). Utilizing primary resistance genes placed into cultivars with enhanced agronomic characteristics, the principal QTL in crop variety has been used to provide quantitative resistance to plant viruses [169]. CRISPR/Cas9 technology has effectively been used to generate virus-resistant crop cultivars and enables the generation of a wider spectrum of CRISPR variations suitable for many applications. However, one of the most popular uses of the CRISPR/Cas9 technology is gene disruption [170], which aids in overcoming the error-prone behaviour of cellular NHEJ (DNA-repair machinery). A frameshift mutation and gene function disruption are brought about by the insertion or deletion (InDel) of nucleotides at sgRNA-targeted locations [171]. By altering the function of the vulnerable gene(s), which changes the plant–virus interaction and reduces viral fitness in the host plant, this technique has been used to engineer virus resistance.

Since viral infections develop quickly and dynamically, managing viral diseases is difficult. By producing viral and non-viral proteins, host resistance (R) genes, and gene silencing via RNA interference, a number of researchers have significantly contributed to the development of resistant plants [172]. The benefits of CRISPR have significantly contributed to the development of plants resistant to DNA and RNA viruses. A. thaliana and N. benthamiana were used in the first experiment to develop CRISPR-mediated viral resistance against yellow dwarf virus (BeYDV) and beet severe curly top virus (BSCTV). Increased resistance to several geminiviruses was seen in tobacco when yellow dwarf virus (YDV) gRNAs coding for replication and cell mobility were overexpressed [173]. Endogenous banana streak virus (eBSV), a double-stranded DNA badnavirus that is a member of the Caulimoviridae family and which inhabits Musa spp., was rendered inactive by the expression of sgRNA that was designed to target the eBSV coding sequence using the CRISPR/Cas9 tool. In comparison to unmodified control plants, the transgenic banana plants exhibited mild symptoms as well as eBSV resistance [174]. The single-stranded RNA (ssRNA) viral genomes were successfully edited using CRISPR/Cas tools, including programmable RNA-guided RNPs such as FnCas9 and CRISPR/Cas13a (LshCas13a; a nuclease from Leptotrichia shahii) [175,176]. Cas13 offers new promise for eradicating dangerous plant viruses because most plant viruses have RNA genomes. FnCas9, a different Cas9 nuclease from Francisella novicida, interferes with plant translation and replication by targeting endogenous RNA [175,177]. CRISPR-mediated immunity against the viruses cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) was developed by expressing gRNAs and FnCas9 in N. benthamiana and A. thaliana, and these plants were observed with a significant reduction in virus accumulation and minimal symptoms. CRISPR/Cas13a-induced genome editing of tobacco Potyvirus and Turnip Mosaic Virus (TuMV) resulted in the development of immunity [176]. Resistance developed in tobacco and Arabidopsis following CRISPR-mediated editing of the pea early browning virus (PEBV) and Tobacco rattle virus (TRV) gRNAs [178]. The Cas13a/sgRNA-expressing transgenic potato plants conferred control of various Potato virus Y (PVY) strains and decreased disease symptoms in potatoes [179]. The LshCas13a system was used to create rice resistant to the Southern rice black-streaked dwarf virus (SRBSDV) and Rice stripe mosaic virus (RSMV) [180]. The benefit of Cas13 for specifically targeting RNA viral genomes in plants needs more research [181]. These research studies show that CRISPR/Cas-mediated targeted viral genome editing is a potent strategy for conferring viral disease resistance in plants.