Advances in Gene Editing Techniques

Abstract

This review highlights advancements in gene editing techniques, including CRISPR-Cas9, base editing, prime editing, PASTE, and modified CRISPR. CRISPR enables gene insertion and substitution by creating double-stranded breaks in the genome using guide RNA. Base editing alters single base pairs, while prime editing corrects transition mutations by adding up to 50 nucleotides with a nickase. PASTE identifies specific genome sites for insertion, adding up to 50 base pairs. These techniques rely on guide RNA and reverse transcriptase to modify the genome. Despite their potential, challenges remain, such as off-target effects, editing efficiency, immunogenicity, and the need for safe delivery systems. Applications include animal breeding, disease treatments (e.g., metabolic disorders, cancer, cardiovascular diseases), immunity enhancement, plant breeding for better traits, and gene therapies. In therapeutics, these techniques also contribute to drug development from biological sources, showing great promise for future medical and agricultural advancements.

Introduction

1. Introduction to Genome Editing:
Genome editing enables scientists to alter DNA sequences, which changes physical traits and can help treat genetic disorders. Techniques use enzymes to cut DNA at specific sites, allowing for the addition, removal, or alteration of genetic material. Major progress occurred in the late 1900s, leading to the discovery of several technologies, with CRISPR-Cas9 becoming the most widely used due to its cost-effectiveness, precision, and efficiency.

2. Key Gene Editing Tools:

A. CRISPR-Cas9:

• Originated from bacterial immune systems.

• Discovered in 1987, gained prominence in 2012.

• Uses Cas9 enzyme and sgRNA to make targeted double-stranded DNA breaks.

• Repairs via NHEJ (error-prone, for knockouts) or HDR (precise, for gene insertions).

• Applications: crop improvement, disease treatment, and functional gene studies.

B. Base Editing:

• Advanced CRISPR tool developed in David Liu’s lab.

• Does not break DNA strands but allows precise single nucleotide substitutions.

• Two main types:

  • Cytosine Base Editor (CBE) – changes C to T.

  • Adenine Base Editor (ABE) – changes A to G.

• Useful for treating single-point mutation diseases (SNPs).

C. Prime Editing:

• Overcomes CRISPR and base editing limitations.

• Allows targeted insertions, deletions, and all 12 types of base-to-base conversions.

• Uses pegRNA and a reverse transcriptase fused to a Cas9 nickase.

• Versions:

  • PE1: Initial version.

  • PE2: Improved efficiency.

  • PE3: Further optimized with nicking for higher success.

• Broad applications in plants and human diseases.

D. PASTE Editing:

• PASTE (Programmable Addition via Site-specific Targeting Elements) can insert large DNA fragments without creating double-stranded breaks.

• Uses integrase enzymes and prime editing techniques.

• Promising for long-sequence insertions in therapy without DNA damage.

3. Challenges in Gene Editing:

• Technical Challenges: Improving PAM specificity, reducing off-target effects, enhancing HDR efficiency over NHEJ, and optimizing delivery methods.

• Ethical Concerns: Ensuring safe, equitable use of genome editing technologies, especially in clinical and agricultural settings.

4. Applications of Gene Editing:

A. Clinical Applications:

• Blood Disorders: Treats conditions like sickle cell disease and beta-thalassemia.

• Cancer: Targets tumor genes and enhances immunotherapy.

• HIV/AIDS: Aims to remove HIV DNA from infected cells.

• Genetic Diseases: Corrects mutations in diseases like cystic fibrosis and muscular dystrophy.

• Viral Infections: Limits bacterial resistance by disabling virulence genes.

B. Disease Treatments:

• Cardiovascular & Metabolic Diseases: Used to modify disease-related genes and metabolic pathways.

C. Plant Development:

• Enhances yield, resistance, and nutritional value.

• Examples include improved rice, wheat, and tomatoes using CRISPR.

D. Therapeutic Applications:

• Corrects genetic defects via gene therapy.

• Enables targeted treatments for genetic conditions.

E. Cancer Treatment:

• Targets and eliminates cancer cells.

• Improves T-cell based immunotherapy.

F. Drug Delivery:

• Nanoparticles enhance targeted drug delivery, with applications in diseases like Alzheimer's.

G. Animal Breeding:

• Produces disease-resistant animals (e.g., pigs, chickens).

• Improves traits like meat quality, milk production, and feed efficiency.

H. Other Uses:

• Sex selection, animal models for human disease research, and hybrid breeding.

Conclusion

Gene editing is an emerging technology with far-reaching consequences across a wide range of fields. Using technologies and tools like CRISPR-Cas9, base editing, prime editing, and PASTE, scientists are opening unprecedented possibilities for accuracy in genetic manipulation. Though there are some limitations, such as ethical issues, technical challenges, and regulatory complexities, the applications of gene editing are significant. These involve improving breeding in animals, promoting disease cure, enhancing plant growth, supporting innovative clinical tests, and advancing therapeutic interventions. As technology keeps developing, it has the potential to revolutionize science, agriculture, medicine, and biotechnology, ensuring a healthier and more sustainable future.

References

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Copyright

Copyright © 2025 Prateek Suthar, Nayan Gupta, Satveer Singh Shekhawat, Parth Dashora, Mr. Aditya Pant, Dr. Bhawani Singh Sonigara. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.