TAG: GS 3: SCIENCE AND TECHNOLOGY
THE CONTEXT: Researchers at the CSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB) and the L.V. Prasad Eye Institute have engineered an advanced genome-editing system.
EXPLANATION:
- This new system modifies DNA with greater precision and efficiency than existing CRISPR-based technologies.
Natural Origins and Repurposing of CRISPR
- CRISPR, originally part of the bacterial immune system, helps bacteria recognize and destroy viral DNA.
- Scientists have repurposed this mechanism to edit genomes in higher-order organisms.
- This innovation, which garnered a Nobel Prize, has significant applications in agriculture, healthcare, and more.
The Off-Target Problem in CRISPR-Cas9
- The CRISPR-Cas9 system, commonly used for gene editing, involves a guide RNA (gRNA) directing the Cas9 enzyme to specific DNA sequences.
- The enzyme then cuts the DNA, allowing the cell’s repair system to modify the genome.
- However, the widely used SpCas9 enzyme from Streptococcus pyogenes often recognizes and cuts unintended parts of the genome, leading to off-target effects.
- Although scientists have developed higher fidelity versions of SpCas9, these often come at the cost of reduced editing efficiency.
Introducing FnCas9 for Greater Precision
- Researchers have explored using the FnCas9 enzyme from Francisella novicida, which is highly precise but less efficient.
- To address this, the team at CSIR-IGIB engineered new versions of FnCas9 by modifying its amino acids to enhance its binding affinity with the PAM sequence.
- This modification allows the enzyme to bind more strongly to DNA, improving gene editing effectiveness.
Enhancing Flexibility and Efficiency
- The enhanced FnCas9, modified for greater flexibility, can access and edit harder-to-reach regions of the genome.
- Experimental results showed that this version of FnCas9 cuts target DNA more effectively than the unmodified version.
- It enhances the scope of CRISPR-based diagnostics and therapeutics by detecting more single-nucleotide changes in the DNA.
Therapeutic Applications and Testing
- The enhanced FnCas9’s suitability for therapeutic use was tested by a team at the L.V. Prasad Eye Institute.
- They edited the genomes of human kidney and eye cells, finding that the modified enzyme performed better than SpCas9 and exhibited negligible off-target effects.
- The team also used this enhanced enzyme to correct a mutation in the RPE65 gene, which causes Leber congenital amaurosis type 2 (LCA2), an inherited form of blindness.
Surprising Efficiency in Treating Blindness
- Researchers isolated skin cells from an individual with LCA2 and reprogrammed them into induced pluripotent stem cells (iPSCs).
- These cells, differentiated into retinal cells, showed normal levels of RPE65 protein after treatment with the enhanced FnCas9.
- The high efficiency and low off-target effects of the edited iPSCs indicated the potential for this technology to treat genetic disorders effectively.
Implications for the Research Community
- The development of an enzyme with high precision and minimal off-target effects is a significant advancement for the research community.
- This precision is crucial for correcting mutations in genetic diseases.
- The next focus for researchers is on developing proficient delivery systems to target cells’ nuclei accurately.
Toward Affordable Therapeutics
- The CSIR-IGIB team is adapting the system for various delivery methods and reducing the size of the enhanced FnCas9.
- Collaborations with Indian companies aim to scale up and manufacture affordable therapeutic solutions for multiple genetic disorders.
- This initiative, driven by indigenous intellectual property, positions India to develop cost-effective therapies for low- and middle-income countries.
