I live in Coimbatore, a city that is called 'The Manchester of South India' because of its extensive textile industry. Although this industry is an asset for our economic growth, our once pristine water bodies, including one whose name ironically means "one who is free of illness", are now greatly polluted by the dye wastewater that the textile factories emit. 

To mitigate this, I have been working on a biological solution. I identified that CRISPR, a revolutionary gene editing technology, can help us target specific pollutants to solve this problem. CRISPR is a gene editing tool that among a range of other applications, allows us to tweak microbes to break down pollutants effectively.  

Here are some basics of this technology:

• Enzymes are biological catalysts that accelerate chemical reactions.

• Laccases are specific enzymes that can break down dyes, like the harmful, carcinogenous Congo Red Dye.

• By using gene editing, we enhance enzyme production in fungi, targeting genes responsible for laccase production. The key gene for our case is lcc1.

• We can think of DNA as a book with a unique story (genetic code) in a language of four letters (A, T, C, G). Occasionally, there are errors (gene mutations) that need fixing. CRISPR is like a team of editors armed with scissors (Cas protein) and sticky notes (guide RNA). They locate the specific gene, cut out the error, and replace it, fixing genetic "typos" and potentially addressing genetic diseases.

Now that we understand the basics, we can dive deep into the step-step-step process of how this works:

1. The CRISPR-Cas9 system is designed by tailoring Guide RNA (gRNA) to attach to the genome, ensuring specificity. The Cas9 protein executes the editing process.

2. A plasmid vector is our genetic toolkit, housing the gRNA and Cas9 nuclease. Components such as origin of replication (ORI), selectable markers, multiple cloning sites (MCS), antibiotic-resistant genes, and restriction enzymes are part of the plasmid.

3. The CRISPR-Cas9 system is introduced into Trametes versicolor through biolistic transformation. This involves the use of a "biological gun" to shoot DNA-coated bullets into the fungal cells.

4. Transformants (cells incorporating foreign genetic material) are identified based on the presence of selectable markers. This ensures the successful integration of the CRISPR-Cas9 system.

5. To confirm the successful editing of the target gene, polymerase chain reaction (PCR) and DNA sequencing are employed on the genomic DNA of selected transformants.

6. RNA-PCR is then used to make numerous copies of a specific piece of DNA called lcc1 cDNA.

7. The lcc1 cDNA is inserted into a vector which serves as a vehicle for carrying the DNA into a cell. The cell produces the desired laccase protein.

8. We then employ enzyme activity detection methods like agar plate assays to confirm the expression and activity of the gene-edited lcc1 cDNA.

I recently spoke about this at an event conducted by a lake restoration organization that I volunteer at, Kousika Neerkarangal (in Tamil that means the water bodies of Kousika, a prominent river in my city). The event was a celebration of water in its most literal sense, as it was named ‘Neeruku Nanri’, which means ‘giving thanks to water’. I am deeply grateful for pure water and for the opportunity to have worked on and shared this project with so many. I look forward to implementing it soon.