The CRISPR/Cas9 system is one of the most extensively researched and developed gene editing tools, primarily consisting of Cas9 and single-guide RNA (sgRNA), which is formed by trans-activating CRISPR RNA (tracrRNA) and CRISPR RNA (crRNA). CRISPR-Cas9 technology has a wide range of applications and holds significant promise for gene function research, the construction of model organisms, and gene therapy, including treatments for cancers, liver diseases, and cardiovascular diseases.

CRISPR-Cas9 Mechanism of Action

Cas9 possesses DNA cleavage domains, including HNH (His/Asn/His) and RuvC (recombinase UVC). The RuvC domain cleaves the opposite strand of double-stranded DNA (dsDNA), while the HNH domain cleaves the complementary CRISPR RNA (crRNA) strand.

Under the guidance of sgRNA, Cas9 proteins can achieve targeted cleavage of specific genes, resulting in double-stranded breaks (DSBs) in the DNA. These breaks are repaired through either cell-autonomous non-homologous end joining (NHEJ) or homologous recombination (HR), leading to the knockout, insertion, or mutational modification of the target genes.

Fig.1 Overview of the CRISPR-Cas9 mechanism of action. (Bora J, et al., 2023)

The specific mechanism of action can be categorized into three distinct stages.

CRISPR/Cas9 Delivery Carriers

Viral Vectors Delivery System

Viral vectors commonly utilized in CRISPR-Cas9 systems include adeno-associated virus (AAV), lentivirus, and baculovirus vectors.

Lipid-Based Nanocarriers Delivery System

Liposome-based nanoparticles (LNPs) are regarded as promising vehicles for the delivery of CRISPR/Cas9.

Polymer-Based Nanoparticles Delivery System

PLGA, chitosan, and other molecules commonly utilized to construct polymer nanoparticle shells enhance the efficiency of cellular uptake of these polymers.

Exosomes Delivery System

Since exosomes directly package sgRNA and Cas9, effectively reducing off-target side effects during transport, they represent promising vehicles for the delivery of the CRISPR/dCas9 system.

Gold Nanoparticle Delivery System

Gold nanoparticles exhibit excellent stability and biocompatibility, and they can be easily modified on their surfaces, making them ideal carriers for the delivery of gene therapies.

Capabilities of CRISPR/Cas9 Technology

Gene Therapy Solutions

CRISPR/Cas9 technology is extensively utilized in gene therapy applications for nucleic acid formulations. It effectively repairs or replaces disease-causing genes through precisely targeted gene editing, thereby addressing genetic disorders. This technology enhances both the efficiency and accuracy of gene therapy while significantly minimizing off-target effects during treatment, ensuring the safety and efficacy of the intervention.

Nucleic Acid Drug Development Solutions

In nucleic acid drug development, CRISPR/Cas9 technology serves as a precise gene-editing tool that can efficiently identify potential drug targets. This technology not only shortens the drug development timeline but also enhances the accuracy of targeted therapies and contributes to the advancement of personalized medicine.

Advantages of CRISPR/Cas9 Technology

• The CRISPR/Cas9 system can quickly identify and cleave specific target genes. This efficiency accelerates the gene editing process, significantly reducing the experimental cycle time.
• CRISPR/Cas9 technology is highly precise in its targeting, effectively recognizing and cutting specific DNA sequences. This exceptional precision significantly reduces the likelihood of off-target effects and ensures the accuracy of the editing results.
• The technology is applicable to a diverse array of organisms and can modify a broad spectrum of gene sequences. Researchers can flexibly adjust the gRNA sequences based on varying experimental requirements to achieve distinct gene editing objectives.

Applications of CRISPR/Cas9 Technology

Fig.2 Applications of CRISPR/Cas9 technology. (CD Formulation)

Gene Function Research

CRISPR/Cas9 technology allows scientists to precisely knock out or modify specific genes in cells and animal models to study the functions of these genes and their associated pathological mechanisms. For instance, by knocking out a particular gene, researchers can observe the physiological changes that occur due to the loss of expression of that gene, thereby elucidating its role in the organism.

Genetic Disease Treatment

Another significant application of CRISPR-Cas9 technology is in the treatment of genetic diseases. By directly correcting disease-causing gene mutations in a patient's genome, these conditions can be treated effectively. For instance, CRISPR has been utilized to correct mutations associated with beta thalassemia and sickle cell disease in in vitro models and preclinical trials.

Microbial Engineering

CRISPR-Cas9 technology has demonstrated significant potential in the field of microbial engineering, particularly in modifying microorganisms through gene editing to produce biofuels, pharmaceuticals, and other valuable chemicals. This technology enables the precise regulation of microbial metabolic pathways, optimizing the generation of desired products.

Development of Biologics

In the field of biopharmaceuticals, CRISPR-Cas9 technology can be utilized to modify genes in mammalian cells, thereby optimizing protein expression and modification during drug production. This capability is especially crucial for the production of biologics with complex structures, such as antibody-based therapies.