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.

Key highlights

• Scientists have developed an enhanced genome-editing system that can modify DNA more precisely and more efficiently than existing CRISPR-based technologies.

• CRISPR occurs naturally in some bacteria, as a part of their immune system that limits infections by recognising and destroying viral DNA.

• 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.