CRISPR: Redefining Precision Medicine

Precision medicine reflects a radical paradigm change in health care by providing targeted treatment based on a person's genetic background, environment, and lifestyle. This differs from standard health care approaches that convey a standard, or "one-size-fits-all" approach, also known as the "trial and error" approach. Precision medicine's aim is to provide the right patient, the right treatment, at the right time. The emergence of high-throughput gene-editing technologies has enabled the push towards precision medicine, and the CRISPR-Cas system has gained unprecedented attention due to its efficiency, precision, and versatility. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) was originally discovered in bacterial genomes as part of their adaptive immune systems, and has since been harnessed as a powerful genome-editing tool. The CRISPR-Cas9 system is the most utilized genome editing system among the different types of CRISPR systems. The Cas9 nuclease is directed to a specific genomic location by a guide RNA (gRNA) and generates a double-stranded break in the DNA. The precise editing provided by CRISPR has proffered additions to the treatment of genetic disorders, cancer treatment, and other rare diseases as part of individualized therapy. Recent developments illustrate the potential of CRISPR not only as a gene -correcting device but also as a platform for cell-based therapies, identifying drug targets, and functional genomics. CRISPR -based platforms have even emerged as acceptable avenues for use in clinical trials aimed at ailments like sickle cell disease, β-thalassemia, and even various cancers. Newly evolving classes of CRISPR, such as prime editing, base editing, and RNA - targeting systems (e.g., Cas13), allow for even greater possibilities not only for gene correction, but also for a more general account of personalization of medicine through safer and more versatile therapy. This review provides a broad overview of underlying mechanisms of CRISPR, investigates CRISPR as a fundamental re-describing of precision medicine, discusses relevant therapeutic applications in disease, pharmacological approaches, delivery, and ethics considerations. Recent developments and ongoing clinical trials with CRISPR underscore how CRISPR is leading a new era for personalized medicine. ^[1-2]

1. CRISPR Technology: An Overview

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a natural defense system that bacteria and archaea use to identify and eliminate invading viral DNA. Recently, scientists have turned this system into a revolutionary gene-editing mechanism that allows researchers to target and alter genomic DNA with precision across organisms, including humans.

The most commonly used CRISPR system is CRISPR-Cas9, which consists of two components:

• Cas9 endonuclease: The endonuclease that makes a double-stranded break (DSB) at the target DNA sequence.
• Guide RNA (gRNA): A synthetic RNA that directs Cas9 to specific site in the genome by complementary base pairing.^[3]

Fig 1: - Mechanism of CRISPR-Cas Systems

• Non-Homologous End Joining (NHEJ): An imprecise, error-prone DNA repair method because it may introduce small persistence or deletions (indels) which can leads to gene knockout
• Homology-Directed Repair (HDR): A more accurate DNA repair method, using donor DNA templates, which could be used to insert specific sequence allowing gene correction or insertion.^[4]
  1. Evolution of CRISPR Systems

An overview of the CRISPR-associated enzymes that allow different types of editing:

• Cas12a (Cpf1): Creates blunt cuts in DNA and requires a different PAM sequence (TTTV instead of NGG).
• Cas13: Can target and degrade RNA which opens up features and applications for post-transcriptional regulation, and sequencing applications for targeting viral RNA.
• dCas9 (dead Cas9): A catalytically inactive form of Cas9 which can be fused to other proteins. This allows you to regulate gene expression and not cut DNA.^[5]

2.2 Advantages Over Previous Gene-Editing Tools

Compared to prior technologies such as Zinc Finger Nucleases (ZFNs) and TALENs, CRISPR has which   includes More straightforward design and programming ,Greater efficiency and value ,Potential for multiplexing (the ability to edit multiple genes at the same time. These advantages make CRISPR an attractive platform for major advancements in both basic research and clinical translation in the field of precision medicine.

2. Role Of Crispr in Precision Medicine

Precision medicine is intended to adjust the healthcare model to each patient based on their own genetic makeup, susceptibility to diseases, and the response to treatment. The CRISPR-Cas system provides an important new tool that addresses this method of personalized medicine directly by allowing for modification of genetic material with precision to edit, regulate, or disrupt, functional human genes and it provides greater accuracy for researcher and scientist to design therapies specific to the patients.^[6-7]

1. 1. CRISPR and Genetic Profiling

One of the mainstays of precision medicine is the understanding of the genetic basis of disease. CRISPR allows for this understanding by the Distinguish gene-disease relationships through knockout or knock-in experiments in cell lines and animal models & To create models that are patient-derived to study the mutations and drug response in diseases like Cystic Fibrosis and Duchenne muscular dystrophy & to find biomarkers through high-throughput CRISPR screens to identify response to treatment or drug resistance.^[8-9]

3.2 Tailored Therapeutic Interventions

CRISPR enables personalized treatments including as the Correction of pathogenic mutations at the DNA level, in many cases for monogenic diseases such as sickle-cell Anemia and β-thalassemia. Enhancement of immune cells (e.g. CRISPR-edited CAR-T cells) and increase in specificity to target cancer cells. Gene regulation therapy using a dCas9-fusion protein to either switch on or off the expression of genes without changing the DNA sequence itself.^[10]

1. 3. Advantages of CRISPR in Precision Medicine

Table 1:- Advantages of CRISPR in precision medicine

1. 4. Real-World Clinical Applications

CRISPR-based precision medicine is already moving into clinical trials:

• CTX001: An ex vivo CRISPR-based therapy for sickle cell disease and β-thalassemia, co-developed by Vertex Pharmaceuticals and CRISPR Therapeutics.
• Edit-101: In vivo CRISPR therapy targeting the CEP290 gene for Leber congenital amaurosis (LCA10), a rare genetic eye disorder.
• CAR-T cell modifications: Using CRISPR to improve immune cell specificity in cancer immunotherapy.^[11-12]

3. CRISPR Applications in Disease Treatment

CRISPR-Cas systems are currently under investigation as therapeutic agents for many different diseases, particularly in therapeutic approaches where standard remedies are ineffective or less specific. Because CRISPR-Cas can correct, silence, or regulating disease-causing genes it is a game-changing tool in the movement towards precision medicine. In this section we will highlight CRISPR's most salient applications in monogenic genetic disorders, cancer, and infectious diseases.^[13]

4.1 Monogenic Genetic Disorders

Monogenic diseases, caused by mutations in a single gene, are ideal candidates for CRISPR-based correction due to their well-defined genetic basis.

• CTX001 by CRISPR Therapeutics and Vertex: A one-time ex vivo CRISPR therapy for β-thalassemia and sickle cell anaemia has shown durable therapeutic response in early-phase clinical trials.^[14]

 4.2 Cancer

CRISPR has numerous possibilities for use in the treatment of cancer through the gene editing of tumoral cells, immune cells and resistance mechanisms.

a. Engineering CAR-T Cells

CRISPR technology is used to knock out immune checkpoints, like PD-1 or TCR genes, in T cells. Enhances the specificity and efficacy of CAR-T cell treatment.^[15]

b. Tumour Suppressor Gene Editing

Correction or deletion of mutations in tumour suppressor genes, like TP53, BRCA1, or KRAS. Takes place mainly in preclinical or animal model studies.

c. Chemotherapy Resistance Reversal

Knock out of MDR1 or other genes related to resistance restores drug sensitivity to chemotherapy.

Current Status: Several trials are testing CRISPR modified immune cells in leukaemia, lymphoma and solid tumours.

4.3 Infectious Diseases

CRISPR represents a new path for targeting viral DNA or RNA, perhaps allowing us to erase a couple of chronic infections from a genomic perspective. CRISPR - based diagnostic systems SHERLOCK and DETECTR enable rapid, sensitive and specific identification of and infectious agent, with proven success identifying SARS-CoV-2, Zika and Dengue viruses. CRISPR-Cas systems (most notably Cas9, Cas12 and Cas13) permitted targeting and cleavage of a viral genome while inside a host cell, which is a possibility to treat chronic infections such as HIV, hepatitis B and herpes simplex virus. Importantly, when it comes to combating antimicrobial resistance, CRISPR has been shown to specifically eliminate drug resistant bacteria by targeting resistance genes, thereby providing a precision pathway to eliminate a superbug. CRISPR also is speeding up vaccine development via precise editing of viral vectors or live-attenuated organisms offering improvements in the development of mRNA and adenoviral vaccines such as those developed during the COVID-19 pandemic. CRISPR also supports functional genomic studies of bacteria or pathogens, allowing researchers to identify essential gene(s) responsible for virulence, replication or even immune evasion that can be exploited for drug discovery. Genome-wide CRISPR in host cells also allows researchers to identify host factors that are essential for pathogen entrance and pathogen replication, which may reveal new possibilities for therapies. While CRISPR technologies are very promising, the use of CRISPR in infectious disease medicine is a concern for several reasons: a possible off-target effect, delivery strategies, immune response issues, ethics issues related to 1. molecular engineered viruses (or other pathogens) and 2. gene editing/gene editing of germline cells. CRISPR-based technologies also permit real-time assessment of emerging infectious diseases. CRISPR has the ability to rapidly identify pathogen mutations allowing for epidemiological surveillance of outbreaks. CRISPR interference (CRISPRi) allows scientists to silence specific genes of bacteria or viruses without cutting the DNA. It can also be used as a method for reversible gene regulation to analyze how pathogens cause infections. CRISPR-Cas engineered bacteriophages are showing promise as next-generation antibacterial agents to selectively kill pathogenic strains, while preserving non-pathogenic microorganisms in beneficial microbiota. In parasitic infection research, such as that studying malaria, CRISPR is being applied to alter the Plasmodium species genomes which will further our understanding of the life cycle, as well as the pathways that promote drug resistance. CRISPR is now enabling gene drive systems which can provide the capability to change mosquito populations under the right environmental conditions by propagating genes that inhibit the capacity of mosquitoes to transduce malaria. Using messenger RNA, researchers are working to apply CRISPR to make "universal antivirals" by using conserved regions of the viral genomes found across strains and species. When studying infectious disease, especially viral processes, CRISPR technologies are generating exciting advances toward dissecting the host immune response to such infections by knocking out immune regulatory genes and using the readouts of these manipulations to better understand the role of these genes in pathogen clearance. High-throughput CRISPR screens in infected human cells also facilitate the discovery of new druggable targets for viral, bacterial, and fungal pathogens. CRISPR is being used to develop personalized approaches in infectious disease treatment with respect to identifying unique patient genetic variants that will guide therapeutics, while accounting for toxicity or effectiveness depending on disease severity. CRISPR's programmability should also enhance multiplexed emerging technologies for detecting co-infections from complex samples, which is essential for diagnosis in resource-limited environments. The promising potential of using CRISPR together with biosensors and microfluidic chips for developing portable diagnostic tools may allow for analysis in remote or point-of-care contexts. Finally, CRISPR technology methodologies also fall under the "synthetic biology" umbrella in their capacity for engineering circuits in immune cells and microbes for infection suppression or immunity in infectious disease contexts. ^[16-17]

Table 3: - Infectious Diseases

Example:

• CRISPR-Cas13 is being used in rapid detection kits (Sherlock, Detectr) for viral RNA, useful in point-of-care COVID-19 testing.

4. CRISPR In Drug Development and Target Validation

The development of effective and safe drugs depends on identifying precise molecular targets and understanding their role in disease. CRISPR technology has emerged as a powerful tool in the drug discovery pipeline, offering unmatched capabilities in target identification, validation, screening, and resistance analysis. It enables researchers to simulate disease models, uncover new druggable genes, and test compound efficacy all with high specificity and efficiency. CRISPR technology allows accurate gene editing to identify genes related to diseases and evaluate previously established targets for therapy. In addition, CRISPR provides new tools for high-throughput genetic screens to systematically knock out the genes and assess phenotypic variations associated with drug discovery. As target validation has a significant cultural challenge, CRISPR provides an additional enhancement for accuracy of confirming a gene's causal relationship in a disease model. Furthermore, CRISPR provides scientists with the ability to generate genetically modified cell lines and animal models that closely represent the human disease for preclinical testing. CRISPRi (interference) and CRISPRa (activation) allow users to study gene function readouts without making permanent changes to DNA, such as  reversible drug-response studies.  By allowing the study of synthetic lethality screening, CRISPR technology is Discovery of relevant gene pairs obligate to cancer cell survival leading to repetitive targeted therapy. In addition, CRISPR technology allows researchers to investigate the mechanisms of drug resistance and the duration of cellular sensitization to drugs by discovering genes that favorably sensitize cells to drugs.  CRISPR will also help therapeutics discover predictive biomarkers useful for stratifying patients for personalized treatment approaches. In addition, CRISPR provides speed and efficiency of linking a genotype to phenotype for the validation of novel drug targets. Pharmaceutical companies are currently developing approaches to use CRISPR technology in drug discovery to help guide and develop improved efficiency so investment can result in new drug development with less risk and consequently improve start-up costs and increase New Drug Application (NDA) success rates. ^[18]

Fig 2: - CRISPR in Drug Development and Target Validation

5.1 Target Discovery and Validation

CRISPR-based gene editing allows for functional interrogation of genes for therapeutic relevance:

• Knockout screens with CRISPR libraries identify essential genes in cancer, neurodegeneration, and autoimmunity.
• CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) tools allow modulation of gene expression without permanent changes, mimicking drug functionality.
• Allow for rapid loss-of-function and gain-of-function studies in a high-throughput format.

Example: CRISPR screens identified PARP1 as a synthetic lethal partner in BRCA1/2-deficient cancers that subsequently led to PARP inhibitor development.^[19]

5.2 CRISPR in Phenotypic Drug Screening

CRISPR engineered cell lines and organoids closely resemble human disease, and therefore drug screening will be more relevant to human pathologies. Enables testing of drugs as applied to patient-derived models that incorporate specific mutations. Screens for drug-gene interactions and can yield predictions for drug effectiveness for patient-specific models. Genome-wide knockout or activation screens are now undertaken through CRISPR as a means to screen for genes that modulate cellular responses to drug treatments. CRISPR utilizes genetic editing of candidate genes to facilitate unbiased phenotypic screens by altering the expression of key genes and subsequently determining any changes to qualitative features associated with morphology, viability, or behavior at the time of exposure to the drug. CRISPR-based libraries have been used to identify genetic modifiers of drug sensitivity or drug resistance in cancer and infectious diseases and neurological disorders. Importantly, CRISPR can be used to identify unknown targets of bioactive compounds by linking genetic perturbations to phenotypic readouts. Furthermore, CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) facilitate fine-tuning of gene expression and study how gene expression changes can produce slight phenotypic changes. Most importantly, CRISPR can be used for multiplexed screening, that is editing several genes simultaneously to study interactions involving multiple drugs and genes. These approaches can uncover pathways that can be targeting using pathway modulator to gene action, and potentially provide investigators with more information regarding genetic networks implicated in the action of any drug. CRISPR enhances phenotypic screening approaches working with patient-derived cells or organoids as this can provide an even greater physiological relevance where drug interactions expect to require sophisticated biological testing. CRISPR can also be used for the additional purpose of improving data collection about off-target effects and toxicities either genetically or pharmacologically by highlighting or abolishing specific genes by retrospectively determine which genes are essential for cell survivability. Therefore, CRISPR adopts rigorous testing methods to provide more intercoupled relationships among cells, drugs, and mechanisms that facilitate new drug discovery whilst also identifying therapeutic targets, biomarkers, and mechanisms of drug action; CRISPR represents a new era in phenotypic drug screening and phenotypic studies.

Fig 3: - CRISPR in Phenotypic Drug Screening

Advantage: Reduces time period to discover new therapies that are guided by biomarkers in cancers, neurological diseases, and infectious diseases.

5.3 Analysis of Mechanisms of Drug Resistance

CRISPR can be used to identify:

• Genes that confer drug resistance (efflux transporters, DNA repair).
• Pathways changed by the therapy that allow moving forward with second-generation drugs.

For example--CRISPR-Cas9 was used to knockout the EGFR mutation variants to describe the mechanism of resistance in non-small cell lung cancer treated with TKIs.^[20]

5.4 Individualized Pharmacogenomics

CRISPR allows us to identify and clarify individuals diversity within their DNA that relates to drug metabolism. As an example, editing variants within genes relating to drug metabolism, such as CYP450 enzyme variants, TPMT, or UGT1A1 could provide tangibles to predict drug metabolism and decrease the concentration of toxic active drug metabolites. CRISPR for use in developing companion diagnostics and safer drug regimens. CRISPR allows for editing of the genes encodes drugs metabolism lines such as CYP450 enzymes making it easier for researchers to model and predict therapeutics for target therapies including adverse drug reaction. CRISPR could also boost personalized medicine by correcting or editing genetic variants in induced pluripotent stem cells (iPSCs), which could then be used to test therapy effects and/or toxicity in vitro. CRISPR-based pharmacogenomic screening can enable researchers to identify genetic mutations affecting drug export and import from tissues to recognize alteration drug targets, transporters or drug metabolizing enzymes and assist in customizing therapies based upon an individuals genetic response formulate therapeutic ideas. With the integration of CRISPR and next-generation sequencing it will easy to validate biomarkers of pharmacogenomics information and deliver customize therapeutics to patients with their condition (e.g. patients with cancer or epilepsy, patients developing cardiovascular problems). To unlimited possibilities, CRISPR allows researcher explore gene-drug interactions from genome-scale data helping them to individually design and administer therapeutics with the least side effects with dosages that are optimized for individuals.

6. Challenges And Limitations of CRISPR in Precision Medicine

Despite the revolutionary potential of CRISPR technology, several scientific, technical, and ethical barriers still limit its widespread use in clinical precision medicine. These challenges must be carefully addressed before CRISPR can be adopted as a mainstream therapeutic modality. ^[21-22]

6.1 Unintentional Effects

One of the great worries is of off-target cleavage, or unintended cuts made by CRISPR-Cas9 at genomic locations, that could potentially lead to:

• Unintentional mutations
• Oncogene activation
• Disruption of essential genes
• Solutions in development:
• Better design of guide RNA (gRNA)
• High-fidelity Cas9 variants (e.g. eSpCas9, SpCas9-HF1)
• Using base editing and prime editing to completely avoid double-stranded breaks

  6.2 Delivery Mechanisms

 Efficiently and specifically delivering CRISPR components to tissues and cells still pose a major challenge. Efficient delivery of CRISPR components is a key barrier to its clinical adoption, and viral delivery systems, including adeno-associated viruses (AAVs), are commonly used because of their ability to transduce cells efficiently, but non-viral delivery systems, including lipid nanoparticles (LNPs), are gaining popularity and providing safer methods of briefly exposing cells to CRISPR machinery while avoiding insertional mutagenesis. Physical systems, including electroporation and microinjection, are useful for ex vivo editing, particularly editing stem cells or immune cells including CAR-T, because they provide more uniform and precise spatial control of gene editing prior to being reinfused back into patients. Recent advances in engineered extracellular vesicles and cell-penetrating peptides are providing exciting new delivery systems for the targeted, non-toxic delivery of CRISPR-Cas systems to tissues or cells. Additionally, more advanced delivery systems using stimuli-responsive and programmable delivery systems, are improving not only the spatiotemporal control of CRISPR activity, but also enhancing the overall infidelity and consequently limiting off-target effects for therapeutic applications. The delivery of CRISPR components in a viable manner is crucial for therapeutic applications involving genome editing. Viral approaches like adeno-associated viruses (AAVs) are ideal because of their high transduction efficiencies and their ability to target specific tissues. Non-viral methods of delivery, such as lipid nanoparticles (LNPs), provide safer, transient expression and are a safer approach for in vivo delivery of Cas9 mRNA and the guide RNA. There is also the possibility of physical methods of delivery: for example, electroporation and microinjection can be used to physically deliver CRISPR reagents inside of the cells of interest, and is often used in ex vivo editing and in vivo animal models. Gold nanoparticles and polymeric carriers have been developed with the hope of increased biocompatibility and biostability, while decreasing immune responses in the case of systemic delivery. New emerging delivery strategies such as cell-penetrating peptides, and exosomes, are proposed innovative delivery platforms that have the ability to deliver reagents in a low-toxicity method, and allow for targeting of uptake in specific tissues. Meanwhile, delivery efficiency is also strongly reliant on the format of the cargo considering that Cas9 can be delivered as DNA, mRNA, or protein (ribonucleoprotein complex), each method having differing stability and expression kinetics. Targeted delivery strategies will employ tissue-specific ligands or antibodies and reduce the risk of off-target effects, maximizing therapeutic precision. Ensuring successful delivery of CRISPR reagents includes challenges such as avoiding immune recognition, nuclear access, and the ability to control the expression of CRISPR components. Overall, improved delivery methods will be a critical aspect for translating CRISPR-based therapies from bench-top to the clinic in a highly safe and efficacious method.

Fig 4: - Types of Delivery Mechanisms in CRISPR