Indian scientists build breakthrough gene-editor, are aiming for patent

• 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.
• CRISPR occurs naturally in some bacteria, as a part of their immune system that limits infections by recognising and destroying viral DNA. In their Nobel-prize winning work, scientists repurposed this bacterial defence mechanism to develop a novel approach for editing the genomes of higher-order organisms.

CRISPR’s off-target problem

• Today, using CRISPR-Cas9, researchers can add, remove, or alter specific DNA sequences in the genomes of animals. This system has been used in various fields, including in agriculture - to improve the nutritional value of plants and increase their yield - and in healthcare to diagnose several diseases and treat genetic disorders.
• The CRISPR-Cas9 gene editing tool uses a guide-RNA (gRNA) designed to find and bind to a specific part of the target genome.
• But the CRISPR-Cas9 system can also recognise and cut parts of the genome other than the intended portion.
• Such “off-target” effects are more common when using the SpCas9 enzyme derived from Streptococcus pyogenes bacteria. Scientists have been able to engineer versions of SpCas9 with higher fidelity but only at the cost of editing efficiency.

Switching SpCas9 with FnCas9

• To overcome these issues, researchers are exploring Cas9 enzymes from Francisella novicida bacteria. While this Cas9, called FnCas9, is highly precise, it has low efficiency as well.
• To enhance it without compromising its specificity, researchers 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,”.
• “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,”.

Juicing the enzyme

• Experiments to measure enzyme activity showed that enhanced FnCas9 cut target DNA at a higher rate compared to unmodified FnCas9.
• CRISPR-based tools for diagnostics and therapeutics rely on the ability of the system to recognise specific single-nucleotide changes in the DNA. Nucleotides are the building blocks of DNA and RNA. Each nucleotide consists of a nucleobase, a phosphate group, and a sugar. Each nucleotide in DNA has one of four nucleobases: adenosine, thymine, guanine, and cytosine. A single-nucleotide change is when just one nucleotide in the genome needs to be “repaired.”
• 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.

A surprising efficiency

• The team was taken aback by the efficiency of the editing. Most of the iPSCs carried the edits, and when the researchers grew colonies from individual edited iPSCs, they found that two colonies showed 100% mutation correction.
• “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,”.