Scientists from the CSIR-Institute of Genomics and Integrative Biology, New Delhi, have developed an enhanced genome-editing system that can modify DNA more precisely and more efficiently than existing CRISPR-based technologies.
To enhance the fidelity without compromising its specificity, researchers led by Debojyoti Chakraborty and Souvik Maiti at CSIR-IGIB modified and engineered new versions of FnCas9.
The researchers tinkered with amino acids in FnCas9 that recognise and interact with the PAM sequence on the host genome. “By doing this, we increase the binding affinity of the Cas protein with the PAM sequence,” Dr. Chakraborty said to the Hindu. “The Cas9 can then sit on the DNA in a stronger configuration, and your gene editing becomes much more effective.”
The researchers also engineered the enhanced FnCas9 to be more flexible and edit regions of the genome that are otherwise harder to access. “This opens up more avenues for gene editing,” Dr. Chakraborty said.
Experiments to measure enzyme activity showed that enhanced FnCas9 cut target DNA at a higher rate compared to unmodified FnCas9.
When the researchers tested the ability of enhanced FnCas9 to identify such changes in the genome, they found enFnCas9 outperformed unmodified FnCas9. An enhanced FnCas9-based diagnostic could target almost twice the number of changes compared to FnCas9, increasing the scope of detecting more disease-causing genetic changes.
Once Dr. Chakraborty’s team had shown the increased efficiency and activity of the enhanced FnCas9 enzyme, a team led by Indumathi Mariappan at the L.V. Prasad Eye Institute in Hyderabad explored the enzyme’s suitability for therapeutic applications.
The researchers used enhanced FnCas9 to edit the genome of human kidney and eye cells grown in lab dishes. It not only edited genes in these cells at a better rate than did SpCas9, it also showed negligible off-target effects.
The team finally sought to understand whether enhanced FnCas9 is a viable option for treating genetic disorders. They tested the enzyme’s efficiency at correcting a genetic mutation that causes Leber congenital amaurosis type 2 (LCA2), a form of inherited blindness. A single mutation in the RPE65 gene results in the loss of expression of a protein called retinal pigment epithelial-specific (RPE65), resulting in severe vision loss.
The team isolated skin cells from an individual with LCA2 carrying the RPE65 mutation, and reprogrammed these cells to become induced pluripotent stem cells (iPSCs). Such cells can be made to grow into any cell type in the human body. When the researchers differentiated the iPSCs into cells of the eye’s retina, the cells expressed negligible levels of RPE65 protein.
The researchers delivered a CRISPR system with the enhanced FnCas9 enzyme into the individual’s iPSCs to correct the mutation responsible for low levels of this protein. When they sequenced the edited cells, they found that the CRISPR tool had corrected the mutation. The edited iPSCs when differentiated into retinal cells also showed normal levels of the RPE65 protein.
Dr. Chakraborty said the team is working on adapting the system to different delivery methods as well as reducing the size of the enFnCas9. “All these will come in the following studies,” he said.
