New Delhi, Dec 10 Scientists from Kolkata-based Bose Institute have created GlowCas9 -- a CRISPR protein that lights up while performing gene editing -- to help boost treatment of genetic diseases and cancer, said the Ministry of Science and Technology on Wednesday.
While CRISPR-Cas9 had been designed to cut and correct DNA with accuracy, scientists could not observe Cas9, the molecular surgeon, in living cells in real time, as traditional detection methods relying on fixing or breaking open cells make it impossible to track the process as it unfolds.
With the newly engineered CRISPR protein, scientists could observe the Cas9 enzyme as it enables them to edit genomes using the CRISPR-Cas9 system for treating genetic diseases, including cancer.
“Gene therapy could be a permanent cure for many life-threatening hereditary diseases. Developing effective, affordable, and safe gene therapy methods remained a challenge for decades,” said the Ministry.
“Tracking gene editing as it happens or watching the molecular machinery as it works, cutting, repairing, and rewriting DNA inside living cells can help monitor CRISPR operations in living cells and tissues without destroying them,” it added.
The finding led by Dr. Basudeb Maji, from the Bose Institute, an autonomous institute of the Department of Science and Technology (DST), opens a new chapter in the visualisation and tracking of genome engineering.
Arkadeep Karmakar, a Ph.D. researcher in Maji’s lab, designed GlowCas9 -- a bioluminescent version of Cas9 that glows inside cells, by fusing Cas9 with a split nano-luciferase enzyme derived from deep-sea shrimp proteins.
“These inactive enzyme pieces reconnect when Cas9 folds correctly, producing light. This is because when the pieces are brought to close proximity, they can reassemble to restore enzymatic activity and produce a visible signal akin to the gentle light of fireflies,” stated the researchers in the study, published in the journal Angewandte Chemie International Edition.
The glowing activity allows scientists to monitor CRISPR operations in living cells, tissues, and even plant leaves --without harming them.
The team found that GlowCas9 is very stable and maintains its structure and activity at higher temperatures compared to the conventional enzyme.
Such sturdiness is important for gene therapy, where stable Cas9 delivery can greatly increase treatment success.
GlowCas9 also increases the precision of homology-directed repair (HDR) -- a DNA repair process crucial for fixing hereditary mutations that are linked to genetic diseases like sickle cell anaemia, and muscular dystrophy.
GlowCas9 can also be tracked in plant systems, hinting at safe, non-transgenic applications in crop improvement, the researchers said.
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