Highlights
- Researchers are exploring Cas9 enzymes extracted from Francisella novicida bacteria (FnCas9) to overcome the issue of the “off-target” effects of CRISPR-Cas9.
- Such “off-target” effects are more common when using the SpCas9 enzyme derived from Streptococcus pyogenes bacteria, which can recognise and cut parts of unintended genomes as well.
- The researchers used enhanced FnCas9 to edit the genome of human kidney and eye cells grown in lab dishes. The enzyme not only edited genes in these cells at a better rate than did SpCas9, it also showed negligible off-target effects.
Breakthrough in CRISPR Technology
Scientists at the CSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB) in New Delhi have made a significant advancement in gene-editing technology. They’ve engineered an improved version of the CRISPR system, which offers greater precision and efficiency compared to existing methods.
This development could have far-reaching implications for both agriculture and medicine.
The research team, led by Debojyoti Chakraborty and Souvik Maiti, focused on enhancing the FnCas9 enzyme derived from Francisella novicida bacteria. Their modifications have resulted in a more powerful and accurate gene-editing tool that addresses some of the limitations of the widely-used SpCas9 enzyme.
Dr. Chakraborty explained their approach: “By tinkering with amino acids in FnCas9 that recognize and interact with the PAM sequence on the host genome, we increase the binding affinity of the Cas protein with the PAM sequence. The Cas9 can then sit on the DNA in a stronger configuration, and your gene editing becomes much more effective.”
Expanding the Scope of Gene Editing
The enhanced FnCas9 (enFnCas9) not only demonstrates improved efficiency but also shows remarkable flexibility. This allows it to access and edit previously challenging regions of the genome, potentially opening up new avenues for genetic research and therapeutic applications.
In laboratory tests, the enFnCas9 system outperformed its unmodified counterpart in several key areas. It exhibited a higher rate of DNA cutting and showed an increased ability to identify single-nucleotide changes in the genome. This enhanced capability could significantly boost the diagnostic potential of CRISPR-based tools.
“The research community needed this precision,” commented Shailja Singh, a researcher at Jawaharlal Nehru University who was not involved in the study. She emphasized the importance of reduced off-target effects, particularly for therapeutic applications aimed at correcting genetic mutations.
Promising Results in Treating Inherited Blindness
To demonstrate the therapeutic potential of their enhanced gene-editing tool, the research team collaborated with scientists at the L.V. Prasad Eye Institute in Hyderabad. They focused on Leber congenital amaurosis type 2 (LCA2), a form of inherited blindness caused by a mutation in the RPE65 gene.
The researchers used skin cells from an LCA2 patient to create induced pluripotent stem cells (iPSCs). They then employed the enFnCas9 system to correct the mutation in these cells. When the edited iPSCs were differentiated into retinal cells, they showed normal levels of the crucial RPE65 protein.
Indumathi Mariappan, who led the team at L.V. Prasad Eye Institute, expressed surprise at the results: “We also examined the whole genome for off-target interactions and found only a few, of no major concern, as compared to several hits seen with other Cas9 proteins [we] examined.”
Towards Affordable Gene Therapies
This breakthrough has significant implications for the development of gene therapies in India and other developing nations. By creating an indigenous, high-precision gene-editing tool, the researchers have laid the groundwork for more affordable therapeutic solutions.
Dr. Chakraborty revealed that the team is already in talks with Indian companies to scale up their technology: “This opens up the doors for not licensing from a foreign entity, which could be very, very expensive.”
Dr. Mariappan echoed this sentiment, stating, “With indigenous intellectual property for such a high precision editor, we are now in a better position to develop newer therapeutics at affordable costs for people in low- and middle-income countries like ours.”
As the research team continues to refine their technology, focusing on improved delivery methods and reducing the size of the enFnCas9 enzyme, the future of gene therapy in India looks increasingly promising. This development not only represents a significant scientific achievement but also a step towards more accessible genetic treatments for a wider population.