Precision Medicine Unleashed: Harnessing CRISPR Technology for Human Health Advancements
Creators: name: Khaliq, Hafiz Muhammad Haseeb
name: Mazhar, Sana
name: Taufiq, Izza
name: Mahmood, Muniba
name: Usman, Muhammad
name: Atta Muhammad, Sumaira
Publisher: name: Zenodo
Year: 2023

description: CRISPR/Cas system is an efficient diagnostic tool that serves as a prerequisite in paving ways for disease therapeutics by providing highly rapid, sensitive, and precise diagnosis as well as epidemiological surveillance. Nucleic acid-based detection strategies exhibit high specificity and sensitivity; however, they require much greater expertise and advanced equipment at a higher cost. CRISPR technique derived from the microbes as a part of their natural defense system forms the basis of gene alteration and has brought a matchless revolution in molecular biology. Moreover, it elicits a very negligible error rate and a prompt diagnosis against the pathogenic sequences of both DNA and RNA nature. This gene editing technique has expanded the tool box of diagnostics by sensing a broad range of targets especially relevant to those that are directly linked to human health so as to provide innovative therapeutic approaches thus curing lethal ailments.

KEY WORDS: CRISPR/Cas, disease therapeutics, epidemiological surveillance, diagnostic toolbox, prompt diagnosis, gene editing

 

 

INTRODUCTION

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a simple, low-cost technique for gene editing that enables the exact modification of genomic sequences to safeguard human health [1]. It is one of the key constituents of a prokaryotic adaptive defense system [2]. These CRISPR systems recognize foreign genetic elements on the basis of their sequence and then with the help of CRISPR-associated (Cas) enzymes, remove these nucleic acids through endonuclease activity [3]. Distinct archaeal and prokaryotic species have different CRISPR-Cas systems, which are linked by their dependence on CRISPR RNA (crRNA), which govern different Cas proteins to recognize and cleave particular targets or sequences of nucleic acid.[4] Through specific binding to a complementary sequence of a protospacer adjacent motif (PAM), crRNA is targeted towards a specific genome [5]. A CRISPR locus is made up of short repetitive sequences that encode tiny non-messenger RNA in between spacers produced from bacteriophages and other extrachromosomal components [6,7]. When a virus attacks the bacteria, new spacers integrate from the genome of the phage, result in adaptive spacers. Different bacteria show specific resistance towards particular spacers which results in inhibition of infection [2, 8]. There are four Cas genes located near the CRISPR locus. There are three phases that constitute adaptive immune system of CRISPR. When a virus or plasmids invade prokaryotes, they develop cellular memory against them [9]. As it in fects, DNA from the invader binds w ith repeating sequences to form a new spacer at the leader end of CRISPR array on host DNA [8] offering particular phage resistance. In expression or crRNA biogenesis, pre-crRNAs, are produced as a result of transcription by RNA polymerase from spacer site of the CRISPR [10, 11].

 

At the upstream of the CRISPR region, tracrRNA is translated in addition to pre-crRNA transcription to perform two important tasks: promoting pre-crRNA maturation of RNase III and DNA cleavage guided by crRNA [12] [13]. Once loaded onto Cas9 i.e., CRISPR-associated nuclease 9, the tracrRNA: crRNA complex creates RNP (ribonucleoprotein complex). In the third step of interference,target nucleic acid is specifically recognized by crRNA guide Cas protein for cleavage. Foreign genetic elements are cleaved through matching CRISPR RNA spacer and target sequence. Due to the sequence homology, the spacer sequence is resent in crRNA [13]. Conjugation of plasmid and resistance in bacteria against phages are mutually beneficial processes that are facilitated by CRISPR and Cas9 [13]. The genome editing system was used to manipulate the genome in diverse cell systems and creaturessince modifying the guide RNA sequence helps to change the target of CRISPR/Cas9 [14]. The CRISPR/Cas system includes 12 subtypes in addition to three primary kinds (types I, II, and III) [15,16]. Single guide RNA (sgRNA) enables Cas9 to select any DNA sequence by modifying the sequence of sgRNA next to the PAM [13]. On the invader DNA that is close to the crRNA-targeted sequence, a short sequence (PAM) of around 2 to 5 base pairs is present [17]. DNA binding and cleavage can be done with the aid of sgRNA and Cutting enzyme (Cas 9) by recognizing these protospacers flankingsites or motifs. For the cleavage of the target sequence, Cas 9 always need the assistance of PAM in addition to sgRNA [18,19]. During the interference process, type I and type II systems of CRISPR also recognized these specific motifs and enhance the activity of cutting enzymes [19,20].

Structural analysis of Cas9 in Streptococcus pyogenes shows that in association with guide RNA and targeted DNA containing PAM form complements. As a result of the formation of hybridization between RNA and DNA, a loop-like structure known as the R loop will form, which resultsin the cleavage of targeted DNA [21]. The NH2 domain of these nucleases results in the cleavage of DNA strands which are complementary to crRNA, whereas the RuvC- like region cleaves the opposite part, resulting in DNA cleavage [18]. Applications for medicating cancer and heritable disorders using CRISPR/Cas9 to fix mutations that are the cause of these diseases. Other single-gene disorders are being treated with CRISPR- based medicines that are still in the pre-clinical stage of research [22]. As compared to the previous gene editing tools, CRISPR is considered to be more beneficial as it is more effective and has the potential to fasten the development of clinical trials [23]. The first clinical use of this technique was done in 2016 when a patient with cancer (lung cancer) was treated with gene-edited cells [24]. Now, the safety of this technology is a major concern, despite the fact that it has great potential for editing of gene. Specific editing mainly broadens the scope of genome editing, while base editors created by combining the Cas9 system with cytidine deaminase can rectify point mutations [25]. In 2020, a new technology known as very fast CRISPR (vfCRISPR) made it possible to create DNA double-strand breaks (DSBs) at nanosecond levels, enabling higher-resolution DNA repair studies in various perspectives [26].
description: CRISPR-Cas MECHANISMS EMPLOYED IN DIAGNOSTICS:

 

CRISPR-based diagnostic strategies are emerging gene-editing tools that revolutionized the science of molecular diagnostics. By minimizing the need of specialized equipment and being highly simple to reconstitute, these molecular strategies are democratizing access to diagnose complex diseases. CRISPR mediated systems provide rapid speed and remarkable precision in detecting the nucleic acids of bacteria as well as viruses. With the revolution of CRISPR technology since the earliest discovery, this gene editing strategy has been expanding enormously so far [27]. These CRISPR systems are specifically characterized into two main classes, with about six types and many subtypes with respect to their evolutionary relationships [28]. On the basis of the RNP effector complex’s nature: Two systems (Class 1 and class 2) have been identified. Class 1 system having multiple effector proteins and Class 2 system with a merely single crRNA binding effector protein. Class 2 systems are primarily involved in disease diagnosis as they comprise the special enzymes showing collateral nature, hence considered to b e building block of complex diagnostic assays. Within the class 1 system, type III effector nucleases named as Csm 6 (also referred as Cas 10) have been engineered to be used in diagnostics by two ways i-e; by combining them with the components of class 2 system or with type III complex (native effector complex).

DETECTR AND SHERLOCK DIAGNOSTIC SYSTEMS:

The two main analogous systems designed for detection of RNA (DETECTR) and DNA (SHERLOCK) are highly precise and sensitive [28]. In the preliminary steps, the clinical samples containing either viral or bacterial sequences are subjected to amplification using RPA (Recombinase polymerase amplification). The amplified fragment is added to CRISPR/Cassystem that first recognizes it, followed by cleavage of that specific sequence (of interest). In this reaction, the Cas nuclease is designed such that it clearly discriminates cleavage only if it getsactivated after it binds to its specific target. Cas 12a is the prominent component of DETECTR and for SHERLOCK, it is Cas 13a. Figure . Two major strategies for CRISPR-based diagnostics; SHERLOCK (Specific High Sensitivity Enzymatic Reporter Unlocking) and DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter)

SIGNIFICANT VIRAL DIAGNOSTIC APPLICATIONS:

Owing to the diverse advantages and remarkable precision, CRISPR acts a promising tool used in the diagnosis of various infectious viral diseases like Zika, Dengue and even Ebola virus [29]. Currently, SARS-CoV-2 detection was carried out employing the DETECTR strategy such that it took 45 minutes time turnaround with the 95% positive whereas 100% negative result prediction in accordance to the PCR mechanism. CRISPR technique can be also applied to carry out detection of clinical samples with the low frequency sequences just like those of AMR (Antimicrobial Resistance) genes by using the NGS high sensitivity protocol in combination.

NON INFECTIOUS DISEASE DIAGNOSTICS THROUGH CRISPR:

In addition to the infectious diseases, the merits of CRISPR advances lie in the detection of non-infectious ailments that are difficult to diagnose. Liquid biopsy protocols are now being operated in combination with the CRISPR technology to examine the tumor ctDNA, EVs (extracellular vesicles), microRNA and other prevailing metabolites in the clinical blood samples which show prominent predictive cancer specific biomarkers of both genetic and epigenetic nature [30].

THE DASH STRATEGY FOR TARGETS OF HIGH FREQUENCY:

The DASH (Depletion of Abundant Sequences to Hybridization) technique basically implies a prior processing stage specifically designed for the high frequency sequences that when subjected to NGS, decrease its sensitivity i-e; mitochondrial rRNA genes in human that are cleaved with CRISPR/Cas system [31]. When these abundant sequences are removed, our targeted desired sequences are subjected to amplification. This emerging technique is now basically used for cerebrospinal fluid (CSF) samples for the amplification of sequences from fungi or parasites with the relatively enhanced sensitivity [32].

FLASH TECHNIQUE FOR LEAST ABUNDANT SEQUENCES:

Another important CRISPR aided strategy known as FLASH (Finding Low Abundance Sequences by Hybridization) mainly blocks all sequences of a particular sample using Alkaline phosphatase activity. The prominent role of CRISPR basically lies in the partial digestion of our targeted sequence in the sample which is then linked to the universal adapters followed by a short round of amplification of the remaining sequences [33]. FLASH combined with NGS identifies the antibiotic resistant genes in aspirates from trachea in the patients infected with the pneumonia by Staphylococcus aureus. When compared to the results of NGS alone, this strategy showed remarkably high fold (5000 folds) targeted gene enrichment. The same protocol was carried out for identification of malarial resistant genes from the dried spot like blood (dried samples) [34]. Astonishingly, CRISPR based FLASH-NGS sensitivity was observed to be very high as that of the NGS alone.

CRISPR-BASED BIOMEDICAL APPLICATIONS FOR TREATING INFECTIOUS DISEASES

Diseases spread through infections, continue to pose a significant public health risk, causing millions of deaths eachyear. Despite the availability of antibiotics, antivirals, and vaccines, the development of drug- unaffectedness pathogens & the slow vaccine pace development has created a need for new approaches to treat these diseases. CRISPR, a revolutionary gene-editing tool, has shown great potential for treating infectious diseases by targeting the genetic material of the pathogen [35]. Gene editing is a potent method for modifying DNA, allowing for targeted cleavage of specific genetic sequences in a pathogen's genome. The capacity to selectively target precise DNA sequences has made CRISPR an attractive tool for treating infectious diseases. CRISPR-based approaches have developed for treating a lot of infectious diseases, including bacterial infections, viral infections, and fungal infections [36]. The advanced technology of CRISPR/Cas9 is highly effective, efficient, accurate, precise and simple. Due to these reasons, it has been employed widely in the research laboratories to experiment on strains of HIV-1 and develop a treatment for AIDS in the recent years. Host factors and genome of HIV-1 are among its targets.

BACTERIAL INFECTIONS: 

CRISPR has proven effective in addressing the issue of antibiotic resistance in bacteria, a significant public health concern resulting from the rise in antibiotic-resistant strains. By targeting and cleaving the genes responsible for antibiotic resistance, CRISPR-based approaches have been shown to restore the effectiveness of antibiotics against drug-resistant bacteria. In addition to targeting antibiotic resistance genes, CRISPR has also been used to target genes involved in bacterial virulence, such as genes involved in the production of toxins or the formation of biofilms [37].

VIRAL INFECTIONS:

 

CRISPR has been utilized to specifically target various viral infections such as HIV, influenza, and herpes simplex virus (HSV). In a particular study, CRISPR was employed to selectively target and cleave the HIV genome within infected cells, resulting in the successful elimination of the virus from those cells. Other than this there is a study in which, CRISPR technique played a vital role in targeting and cleave the genome of the influenza virus, preventing the virus from replicatingCRISPR has also been used to target the HSV genome in infected cells, which could potentially lead to the development of a cure for HSV infections [38].

 

FUNGAL INFECTIONS:

CRISPR has been used to target genes involved in fungal virulence, such as genes involved in the production of virulence factors or the regulation of fungal pathogenicity. For instance, CRISPR has been cast-off to target and cleave the genes responsible for virulence in Candida albicans, a common fungal pathogen. In addition to Candida albicans, CRISPR has also been used to target other fungal pathogens, such as Aspergillus fumigatus and Cryptococcus neoformans. CRISPR-based biomedical applications have shown great potential for treating infectious diseases. By focusing on the genetic material of pathogens, CRISPR exhibits remarkable potential for treating diverse infectious diseases with precision and efficacy. Although CRISPR-based strategies are currently in the early stages of advancement, they offer immense prospects for the future of infectious disease treatment.

HIV (HUMAN IMMUNODEFICIENCY VIRUS):

HIV causes a lethal disease AIDS (acquired immunodeficiency syndrome) which is recognized as a dreadful problem throughout the world. AIDS has become an incurable and chronic disease because the highly active antiretroviral therapy (HAART) is unable to effectively remove viral reservoirs that are latent [39]. However, this therapy reduces the death rate of patients significantly [40]. There is an urgent need for developing therapies to eliminate viral latent reservoirs and inhibit the replication of HIV. The major gene editing tools based on nuclease-mediating editing include three mechanisms which are; ZFNs also known as zinc finger nucleases, transcription activator-like nucleases i.e. TALENs and CRISPR-associated nuclease 9 (Cas9). These strategies have been widely used in researching for treatments for HIV/AIDS [41]. A successful clinical trial has been conducted on patients infected with HIV-1 using ZFNs-CCR5 (chemokine receptor type five) with CD-4 for T-cells editing. It demonstrated that modifications in ZFN-CCR5 may prove to be safe and effective in treating AIDS in humans [42].

GENE-EDITING TOOLS:

Three stages of HIV infection are prevalent in infected patients which are acute, chronic (latent) and clinical disease i.e. AIDS [43]. The envelope protein gp120 of HIV-1 fuses with the CD4 receptor and then binds with the co-receptor CCR5, present on the membrane of the cell. The most modern technique for gene-editing that has been developed recently comprising of several advantages of deleting, inserting and replacing target DNA sequences named as CRISPR/Cas9 [44]. ZFNs and TALENs are expensive and time-consuming techniques whereas, CRISPR/Cas9 is an accurate and rapid methodology for preventing infection by HIV-1 [45]. Many studies have been described to report the potential advantages of CRISPR/Cas9 gene editing for therapy of AIDS and prevention of HIV [46].

CRISPR/CAS9 MEDIATED GENE EDITING:

Recently researchers demonstrated that in vitro target DNA cleavage may be introduced by an endonuclease named Cas9 directed by guide RNAs [47]. Since then, gene therapy for human CD34+ hematopoietic stem and progenitor cells (HSPCs) has advanced greatly because of the vast uses of CRISPR/Cas9 technology [48]. The Cas9 helicase, which is part of the CRISPR-Cas9 system, may attach with RNA synthesized after cleaving host DNA consisting of palindromic repeats and cut out invading RNA spacers linked to DNA. These short fragments of host DNA are obtained from additional elements of chromosomes. In addition to efficiently facilitating gene editing, CRISPR/Cas9 technology can also provide biological benefits. A mutation present in two nuclease domains of Cas9 inactivates it, resulting in dCas9, that is employed as a DNA-binding protein that is locus-specific [49]. This dCas9 can control expression of genes when it is fused to a transcription activator or repressor molecule [50]. Additionally, new gene editing methods have been created on the basis of CRISPR/Cas9 technology, including CRISPR/Cpf1, Cas13a/C2c2, an endonuclease without tracrRNA and an RNA-targeting effector that produces cleavage in ssRNA [51]. As a result, CRISPR/Cas9 is a formidable technique for editing of genes with limitless utilization in the realm of biomedical sciences.

TREATMENT OF HIV/AIDS BY APPLYING CRISPR/Cas9 TECHNOLOGY:

Targeting latent viral reservoirs effectively is the fundamental challenge to HIV/AIDS therapy. HAART is a therapy which can slow down replication of HIV-1 in patients but still it is unable to completely remove the provirus present in resting CD4+ T cells [52]. As a result, reservoirs consisting of latent viruses can persist for several years. In earlier study, the HIV-1 LTR was particularly targeted by Tre-recombinases, which led to the removal of HIV-1 virus particles from HeLa cells. Additionally, Qu et al. produced ZFNs that specifically targeted the TAR region of the HIV-1 LTR, which effectively excised off the integrated pro-viral DNA of HIV-1 from T cells and the latent cell line C11 [53]. In 2013, the gene editing strategy based on CRISPR/Cas9 was first demonstrated for the treatment of HIV/AIDS. By focusing on the long terminal repeats (LTRs) of HIV-1, Ebina et al. were able to successfully employ CRISPR/Cas9 to restrict the expression of genes in Jurkat cell lines. The locations targeted were NF-B binding cassettes in the U3 region of LTRs and sequences known as TAR present in the R region [54]. As a consequence, the replication of the HIV-1 provirus and production of RNA transcripts were effectively inhibited. Moreover, it was evaluated that CRISPR/Cas9 could inactivate viral genes integrated internally by removing them from the infected host cell genome, showing that it could be effective in the treatment of HIV/AIDS. Furthermore, studies were done and experiments were performed to assess the HIV-1 genome and how it can be removed by utilizing CRISPR/Cas9. According to advanced research on this technology, CRISPR/Cas9 can cut non-integrated sequence of HIV-1 that will result in three to folds decrease in the integration of HIV-1 pro-viral DNA. Surprisingly, the nonintegrated HIV-1 provirus reacts to the DNA repair pathway mediated by NHEJ [55]. As a result, the CRISPR/Cas9 acts in both HIV-1 and pro-viral DNA in dormant cells, which enhances its accurate ability and potential to develop a cure for HIV/AIDS.

APPLICATIONS OF CRISPR IN CANCER TREATMENT:

In order to treat or cure disorders, a medical technique known as somatic gene therapy involves inserting new genetic material into non-reproductive cells. Although this technique has been in use since the 1980s, there have been significant drawbacks because of issues such gene silencing, host immunological reactions, and unintentional effects on untargeted genes or tissues. However,recent developments in gene delivery technologies, such as viral vectors, as well as a deeper comprehension of immune responses and gene regulation have resulted in the creation of safer and more efficient gene treatments. Currently, hereditary illnesses and specific forms of cancer can be treated using gene treatments that have received FDA approval.[56]. In 2014, zinc finger nucleases (ZFNs) rather than CRISPR/Cas9 were utilized in the first clinical experiment for gene therapy to treat HIV. Patients with persistent HIV viremia participated in the experiment and Underwent treatment with potent antiretroviral drugs medication along with CD4+ T cells that hadbeen genetically altered with ZFN. The trial's findings revealed that the majority of patients had lower blood levels of HIV DNA [57].

Gene therapy is significantly hampered by the delivery of nucleases like Cas9, ZFN, and TALENs. All nucleases encounter comparable difficulties when introduced into human cells, despite the fact that they are biomacromolecules with various characteristics. The main exception is that the CRISPR/Cas9 system requires the guide RNA (gRNA) to be given as DNA or RNA molecules. These nucleases may often be administered as DNA, mRNA, or protein.[58]. The in vivo delivery of CRISPR/Cas9 is more efficient and attractive when done using viral vectors. CRISPR/Cas9 can enable permanent genome editing with a single treatment of transitory expression, in contrast to conventional non-integrated viral-based gene therapy, which necessitates continual transgene expression via repeated dosages. The severe requirements for viral vectors in delivering CRISPR/Cas9 may be lessened by its high efficiency, specificity, and stability.[59]. Mostly used are IDLV [60] and AAV [61].

 

ADVS AND AAV VECTORS:

Utilizing viral vectors improves the effectiveness and allure of CRISPR/Cas9 in vivo delivery. Contrary to conventional non-integrated gene therapy utilizing viral vectors, which requires ongoing transgeneexpression via repeated doses, CRISPR/Cas9 can enable permanent genome editing with a single treatment of transitory expression. The great efficiency, specificity, and stability of CRISPR/Cas9 may mitigate the stringent requirements for viral vectors in its delivery.[62] Host inflammatory responses, especially cytotoxic T cells, restrict the utilization of AdV vector[63].

LIVER CANCER:

CRISPR/Cas9 has been utilized to specifically focus liver cancer cells, with encouraging results in preventing metastasis and growth. A particular sgRNA has been developed to specifically target the NSD1 gene in HCC cells, which controls the Wnt/-catenin signaling pathway linked to the development of liver cancer. By suppressing the expression of Wnt10b, CRISPR/Cas9-mediated NSD1 KO cells demonstrated decreased proliferation, migration, and invasion in vitro and in vivo. According to these results, CRISPR/Cas9 may be able to stop liver cancer from progressing by inhibiting the Wnt/-catenin signaling axis.[64,65] The Wnt/-catenin pathway is adversely regulated by CRISPR/Cas9, which results in decreased movement, invasion, and proliferation. This can be used to impede the advancement of HCC [65-70].

 

CERVICAL CANCER:

Ling et al. (2020) demonstrated that p53 and p21 protein levels in cervical cancer lines (HeLa and SiHa) and tumor cell-bearing mice were enhanced by simultaneous targeting of both E6 and E7 [71-74]. It has been suggested as a potential cervical cancer treatment plan that HPV E6 causes When E6 is removed, p53 is inactivated in tumor cells, then reactivated, and p53 expression and signaling are transmitted.[75,76]. By using gene editing techniques to target oncoproteins E6 and E7 in HPV16 and HPV18, it may be possible to deactivate these oncogenes, causing cell cycle arrest and death.
description: CONCLUSION:

The progress of CRISPR as a gene editing tool has changed the field of life science in completely a new way where the compatibility and versatility of this system has been increasing day by day. And have generated potent new ways for analyses or study of various biological systems and human disorders. The targeted modification of transcriptomes, genomes, epi-genomes, and nucleic acids bioimaging, the documentation of biological activities, and the detection of genetic material are few of the uses that CRISPR-Cas systems have been put to thus far. Aiming to develop diagnostic tests for genomic-material based point-of-care (POC) for use in typical clinical treatment, the rapidly developing field of diagnostics relies on the specificity, versatility, and simplicity of CRISPR technology. The dependence of the majority of current diagnostics methods of CRISPR on pre-amplification for the identification of targets below the femto-molar range is a significant drawback.

Although the primers used for preamplification boost specificity by a further level, this method complicates the process, makes its costly, and lengthens its time of reaction. Improvements could be made by using signal-amplification techniques that do not rely on primers or by altering the Cas enzyme, crRNA, and different reporter molecule. Most of the crRNAs and primers for pre- amplification must currently be tested, and form guidelines for elimination of viable possibility.

CRISPR-based diagnostics allows patient-centric with DNA or RNA based method of diagnostics to separate patients who are going to get good from the medicine from those who are at risk for major side effects and to track treatment responses to a drug. Additionally, by detecting cell-free mRNA, CRISPR-based diagnostics may be utilized to track gene expression in real-time across many organs. Future uses of CRISPR-based diagnostics will certainly entail materials science- related technologies for the detection of genomic material on surfaces of solid, in wearable technology, or in different medical equipment.
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