CRISPR/Cas9 Gene Editing Technologies in the Development of Disease Therapies

What are the Gene Editing Technologies?

Gene editing is a branch of genetic engineering, which is a breakthrough method capable of inserting, deleting, modifying, or replacing DNA in the living genome. Gene editing technology is a technique that targets specific changes in genetic material. In recent years, the successive emergence of Zinc Finger Nucleases, Transcription Activator-Like Effector Nucleases (TALEN), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), and Base Editors (BE) has not only provided powerful tools for gene function research but also offered new therapeutic strategies for life sciences and medicine. Gene editing technology has been widely applied in the construction of animal cell models, drug target screening, gene function research, and holds broad prospects in the field of gene therapy.

Conceptual diagram of gene editing.

What is CRISPR/Cas9?

The CRISPR/Cas9 system is composed of the Cas protein, which possesses nucleic acid endonuclease activity, and a single-stranded guide RNA (gRNA) containing a artificially recombined segment of the target gene. The gRNA can direct the Cas protein to perform knockout, insertion, and mutation modifications on the target gene. The CRISPR/Cas9 system generally exists in three different biological forms: plasmid DNA (pDNA), RNA, or Cas9 ribonucleoprotein (RNP). CRISPR-Cas9 technology has broad applications in gene function research, model organism construction, gene therapy, including cancer, liver diseases, cardiovascular diseases, etc., showing promising prospects.

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Application of CRISPR/Cas9 Gene Editing Technology in Virus Research

In the field of infection and research of pathogenic viruses, CRISPR/Cas9 gene editing technology enables the precise editing of target genes by specifically identifying the location of viral genomes or host target genes through gRNA, prompting Cas9 nucleases to accurately cleave complementary double-stranded DNA, thereby achieving the effect of antiviral therapy.

Novel Coronavirus Research

In the prevention and treatment of novel coronavirus, CRISPR/Cas9 gene editing technology can identify key genes affecting virus replication and find drug targets through these genes, facilitating drug development.

• Using CRISPR/Cas9 screening methods focusing on ISGs to identify restriction factors of SARS-CoV-2, screening validation results suggest that DAXX is an effective antiviral factor restricting SARS-CoV-2 replication.
• Whole-genome gene knockout screening of CRISPR/Cas9 was performed in human lung epithelial cell line Calu-3 to identify host factors essential for SARS-CoV-2 virus infection and survival, and the study discovered that GATA6 is a pre-viral host factor for SARS-CoV-2.

These findings provide new insights for targeted treatment of novel coronavirus infection, and CRISPR/Cas9 gene editing technology can also play an important role in rapidly identifying new pathogens.

HIV Virus Research

In the research and treatment of HIV, CRISPR/Cas9 can serve as a defense mechanism by guiding RNA (gRNA) and Cas nucleases to bind to complementary sequences in the invading viral nucleic acids and degrade them.

• By introducing Cas9 protein and antiviral gRNA directly targeting the viral genome in HIV-infected cells, HIV virus replication can be suppressed. However, due to acquired mutations around Cas9 cleavage sites, some viruses may evade this suppression. Therefore, combination therapy of CRISPR/Cas9 gene editing technology with other anti-HIV drugs is more effective than standalone CRISPR/Cas9 gene editing technology treatment.
• Additionally, using continuous long-acting slow-effective release antiviral therapy (LASER ART) alongside CRISPR-Cas9 treatment has resulted in the clearance of virus from the latent infectious reservoir of HIV-1 infected humanized mice, demonstrating the potential for eradicating HIV-1 virus completely.

HBV Virus Research

In the research and treatment of HBV, as HBV genetic material is covalently closed circular DNA (cccDNA), current antiviral therapies cannot cure chronic hepatitis B virus (HBV) infection. Thus, CRISPR/Cas9-mediated cccDNA-specific cleavage is a potential curative strategy for chronic hepatitis B (CHB).

• Using adeno-associated virus (AAV) vectors and CRISPR-Staphylococcus aureus (Sa) Cas9 to edit chronic HBV-infected and entecavir-treated humanized mice liver genomes, it was found that HBV-specific AAV-Sa Cas9 therapy significantly improved the survival of human liver cells, showing a trend of reduction in total liver HBV DNA and cccDNA, and good tolerability.

This work provides a basis for the feasibility of CRISPR/Cas9 therapy or even cure of chronic HBV infection, albeit with risks of genome rearrangements and damages. Utilizing CRISPR/Cas-mediated base editors (BEs) to permanently inactivate the HBV genome without cutting DNA opens up a new avenue for Cas9-BE curing HBV.

Applications of CRISPR/Cas9 Gene Editing Technology in Neurodegenerative Diseases Research

In the field of neuroscience, the CRISPR/Cas9 technology is not only applicable to in vitro neuronal cells but can also be employed in fertilized eggs, embryos, and during adulthood to elicit effects at the level of in vivo brain tissues. Its applications encompass brain gene and function studies, construction of gene knockout/knock-in mouse models, and experimental therapies for certain neurological disorders.

Huntington's Disease Research

Huntington's disease (HD) is an autosomal dominant, progressive, hereditary neurodegenerative disorder caused by a segment of CAG repeat sequences in the HTT gene. The mHTT protein forms aggregates in brain cells, leading to neuronal death.

• Studies have reported the analysis of mHTT expression mediated by CRISPR/Cas9 in the striatum of HD 140Q-KI homozygous mice and the behavioral capabilities after CRISPR/Cas9 clearance of mHTT in heterozygous mice, confirming that CRISPR/Cas9-mediated gene inactivation can reverse neuropathological features and behavioral phenotypes, effectively mitigating HD-related phenotypes. The common method for implementing brain CRISPR/Cas9 gene editing in adult mice is through AAV virus mixture injection, although the long-term efficacy and safety of this technique still require rigorous testing.

Alzheimer's Disease Research

Alzheimer's disease (AD) is an irreversible, progressive neurodegenerative disorder characterized by cognitive impairment and the presence of amyloid-beta (Aβ) plaques and neurofibrillary tangles.

• Studies have reported in vivo neuronal gene editing using CRISPR-Cas9 amphiphilic nanocomplexes, targeting Bace1, which inhibited pathological and cognitive deficits associated with Aβ in two Alzheimer's disease mouse models. Through observations using Sanger sequencing, whole-genome sequencing (WGS), and whole-exome sequencing (WES), it was found that this system not only significantly downregulated Aβ42 plaque aggregation in Alzheimer's disease model mice but also minimized off-target effects. This research utilizing Cas9-nanocomplexes without viral vectors for in vivo gene editing broadens the potential applications of the CRISPR/Cas9 system in many neurodegenerative diseases.

Compared to other therapeutic methods requiring continuous interventions, CRISPR/Cas9 can permanently eliminate the expression of target genes. Moreover, the severe neurological symptoms of many neurodegenerative diseases are associated with the preferential vulnerability of selective neuronal populations. Selecting specific promoters can allow CRISPR/Cas9 to act specifically. Therefore, the use of CRISPR/Cas9 in the research and treatment of similar neurodegenerative diseases holds promising prospects.

Application of CRISPR/Cas9 Gene Editing Technology in Cardiovascular Disease Research

Currently, many types of heart diseases are often associated with single gene mutations, some of which are transmitted in an autosomal dominant inheritance pattern. Clinical treatments often focus on disease management without addressing underlying genetic defects. Utilizing CRISPR/Cas9 technology to construct animal models of heart-related diseases and scientifically investigating the mechanisms of disease occurrence, as well as exploring gene therapy research, will bring tremendous benefits to clinical cardiovascular patients.

Coronary Heart Disease Risk Factors Research

Coronary heart disease (CHD) is essentially a complex organic heart disease caused by coronary artery atherosclerosis leading to coronary insufficiency, which involves genetic and acquired environmental factors. Previously, PCSK9 edited with the CRISPR/Cas9 system has been validated at the animal level in mouse models. The results indicate that the targeted gene efficiently expresses in the adult liver, significantly reducing blood PCSK9 levels, and lowering blood cholesterol levels by 40%. Additionally, CRISPR/Cas9 has been demonstrated to effectively target human PCSK9 in human liver cells in vivo in a liver-humanized mouse model, suggesting that genome editing therapy targeting PCSK9 is similarly effective in humans. Furthermore, the APOC3 gene has been identified as an important new target for reducing cardiovascular disease. If gene editing technology can control coronary heart disease risk factors at the genetic level, it will revolutionize the era of traditional drug prevention of coronary heart disease.

Arrhythmias Research

Arrhythmias are a common group of diseases in cardiovascular diseases, which can occur independently or in conjunction with other cardiovascular diseases. Hereditary arrhythmias are mostly caused by gene channel mutations, such as long QT syndrome, short QT syndrome, Brugada syndrome, etc. Although many pathogenic genes have been discovered, it is difficult to predict the pathogenicity of individual mutations and the clinical consequences thereof, posing significant challenges in clinical diagnosis and treatment. Cardiomyocytes derived from induced pluripotent stem cells (iPSCs) induced from Brugada syndrome patients have been corrected using CRISPR/Cas9 technology to correct the SCN5A deletion mutation in patient iPSCs, improving Brugada syndrome-related phenotypes at the single-cell level, including restoration of electrical characteristics and calcium handling ability. Overall, the derivation of iPSCs from patients and the establishment of genetically engineered human models using CRISPR/Cas9 have significant potential to advance research on arrhythmias and provide promising treatment strategies for hereditary heart diseases.

Application of CRISPR/Cas9 Gene Editing Technology in Gene Therapy Research

Currently, a large number of CRISPR/Cas9 gene editing studies involving various species in vitro and in vivo have demonstrated the enormous potential of this technology, bringing hope for disease treatment research and clinical applications based on this technology. Based on the non-homologous end joining and homology-directed DNA repair mediated by CRISPR/Cas9 gene editing technology, several recent studies have successfully applied this technology to repair genetic defects associated with genetic diseases, including point mutations and genomic deletions.

Application of CRISPR/Cas9 Gene Editing Technology in Cancer Research

Currently, CRISPR/Cas9 gene editing technology has been applied in various aspects of cancer research, including functional studies of tumor-related genes, construction of animal tumor models, screening of tumor cell phenotypes and drug resistance-related genes, and gene therapy for tumors.

Enhancing the Expression of Tumor Suppressor Genes Using CRISPR/Cas9

Tumor suppressor genes can promote cell differentiation and inhibit excessive proliferation and migration of cells. It has been reported that PTEN (phosphatase and tensin homolog) possesses phosphatase activity, which dephosphorylates PIP3 (phosphatidylinositol trisphosphate) to PIP2 (phosphatidylinositol-4,5-bisphosphate), thereby inhibiting the PI3K/Akt (phosphatidylinositol 3-kinase and protein kinase B) signaling pathway, ultimately inhibiting tumor occurrence and proliferation.

Correcting Harmful Mutations Using CRISPR/Cas9

As an epigenetic modifier and tumor suppressor gene, ASXL1 (additional sex combs like 1) mutations are present in chronic myeloid leukemia (CML) and are associated with poor prognosis. In the leukemia cell line KBM5, a nonsense mutation in ASXL1 occurred, leading to the absence of ASXL1 protein expression in KBM5. Studies have used CRISPR/Cas9 gene editing technology to target the repair of ASXL1 nonsense mutations, resulting in re-expression of ASXL1 protein and restoration of PRC2 (polycomb repressive complex 2) function. Significant reductions in cell growth and increases in myeloid differentiation were observed in ASXL1 mutation-corrected cells. This study demonstrates the feasibility of using CRISPR/Cas9 for gene correction.

Targeting Knockout of Cancer-Causing Genes Using CRISPR/Cas9

It has been reported that six sgRNAs targeting ORFpreS1/preS2/S were designed and the sgRNAs were constructed into CRISPR/Cas9 expression vectors, resulting in HBs Ag knockout liver cancer cell lines.

Prospects for Gene Editing Technology

Rapid advances in gene editing technology have dramatically increased the ability to make precise alterations to eukaryotic genomes. Editable nucleases, especially the CRISPR/Cas9 system, which is more efficient, simple, and low-cost for editing, have revolutionized the study of genome function. In addition, the emergence and continuous improvement of single-base editing technology has enabled the precise conversion of individual bases and reduced off-target efficiency. Although gene editing technology is still facing the problems of off-target efficiency and potential side effects such as immune response, with the cross integration of multiple disciplines in the future, the new generation of gene editing technology will surely be easier, more efficient and more precise.