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‘Playing God’ with CRISPR

Updated: Aug 22, 2022

Sundaram Acharya

Today is 7th October 2070. I am going out with my colleagues to celebrate the golden jubilee of CRISPR Nobel Prize. My granddaughter, Neeri is accompanying me. She is just 8 years old, but her inquisitiveness is much beyond that of children of her age.

Neeri, “Dadda, what are we celebrating today?”

“Two women scientists, Dr Jennifer Doudna and Dr. Emmanuelle Charpentier. They had won the Nobel Prize in chemistry for discovering CRISPR genome editing technology 50 years ago and this discovery put mankind at a unique time in history.”, I tell her.

Neeri, “Wow! How does it change the world?”

“Well, in a broad sense this discovery has given a boon to mankind to play God, the unbounded ability to play with our evolution and fate.”

She grins with glee at this comment and exclaims “Modern day Prometheus!”

I laugh at her saying, “Reading lots of Greek mythology, huh!”

Then start explaining, “CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats. This system is a result of millions of years of arms race between bacteria and viruses. It gives bacteria the power to thwart their enemies, the viruses or bacteriophages. You can imagine it as a bacterial equivalent of the human immune system which prevents us from getting viral flus, bacterial infection and so on. Even the yogurt bacteria that scientists call ‘Streptococcus thermophilus’ has this system within.’

She couldn’t resist to say, “I like yogurt!”

“That's why you're so interested in CRISPR!” I grin.

“How CRISPR is a big boon to humanity?” she asks.

“Because CRISPR can cure any genetic diseases; It can detect viruses; It can make food tastier and more nutritious; and many other tempting things that mankind has aspired to achieve for so long...” She chimes in, “But, how?”

I reply, “Do you know why your granny always insists that you should eat lentils?”

And with this question I could sense a speed breaker in her excitement. In her distracted voice she replies, “Because they have proteins and proteins are building blocks of life.”

I ask, “Have you ever wondered where these proteins come from?”. And again, her excitement is in full acceleration.

I dive into explanation due to her contagious enthusiasm, “You know, our body is made up of trillions of cells and every cell has a nucleus. In the nucleus, there is a code which has six billion letters.”

Before I could say more, she screams on top of her voice “That's DNA!” She is not only excited but so confident with her answer.

I smile at her and continue, “Yes! We call it DNA and it is written with only four letters, A, T, G, and C. The DNA gives instructions to our cells to make proteins via an intermediate RNA. Cells use these proteins to get their message across which is why proteins are considered a cell's building blocks. A single misspelled letter in the DNA can lead to a life-threatening disease due to a faulty protein being produced. In the computer assignment you have written on Microsoft word, imagine you have misspelled in some places. You can easily correct it by replacing the wrong letter with the correct one. Likewise, the word file of DNA can be edited through CRISPR.”

After a long pause, she asks, “How to find a single letter out of so many? Is it not similar to finding a needle in a haystack!”

I add, “Not only finding, it's finding and changing. CRISPR can do this with unparalleled ease.” She exclaims, “But how?!”

“Well, it's a long story though.” I laugh.

And start explaining, “At the heart of CRISPR-Cas9 system lies two components, a Cas9 enzyme, and a guide RNA (gRNA). Imagine, Cas9 is a car travelling on a track of DNA with gRNA as its’ steering wheel. RNA is like a chemical sister of DNA. It carries four letters too, just the T is replaced by U. Just like you get along well with your best friend, RNA does the same with DNA. That’s why the two can be paired up so easily. This we call as base complementarity.”

“It’s like a family.” she laughs.

“Not only family, they kinda have their own society!” I continue, “Just like your sister is glued together with her best friend, DNA too unites with another piece of DNA. This is what scientists refer to as double stranded nature of DNA. The two strands of DNA should get separated for a gRNA to couple with the DNA. The Cas9 enzyme powers this mechanism and upon DNA-RNA coupling, it cuts the two strands of the DNA, which is known as a double-strand break (DSB). Just like you paste via Ctrl+V in a Microsoft word document, Cas9 can paste a correct letter in the cut position, thereby editing the misspelling. Scientists name this homology directed repair (HDR) if I go by the scientific jargon.”

With great excitement, she asks “It's like a fairy tale!”

I share a smile seeing her excitement and add, “As they say, ‘Not all fairy tales have happy endings’. The same happened here as well.’’

“Why, what is the problem?” she asks in a quavering voice.

I continue, “The code of gRNA should match that of DNA which is to be cut by Cas9. Like in your class, every student is not equally careful in their studies, Cas9s are not all careful in reading the code that matches between gRNA and DNA. The SpCas9 from bacteria Streptococcus pyogenes is notoriously careless and can lead to cutting of DNA at unintended places anywhere within the six billion letters that constitute the human genome. This is called off-targeting and can result in debilitating diseases. That's why scientists have been trying to find other alternatives to SpCas9 similar to how your class teacher appreciates a relatively better, more careful student over others. During that period, I was doing my PhD research on CRISPR technology to address the problem of off-targeting and our team found an alternative...”

“What alternative did you find?” she gushes over before I could even complete.

“That’s a story very close to my heart. Our team discovered a Cas9 protein from a different bacterial species, Francisella novicida (FnCas9), which was found to cut DNA only when DNA and gRNA code-matching is perfect. This we call as specificity.”

“That means when imperfect matching happens, FnCas9 does not cut DNA so, ‘that’ targeting cannot happen.” She answers. “Off-targeting, right?”

“Exactly so! I am happy to see that you have understood ‘off-targeting’. Our team developed a technology, FnCas9 Editor Linked Uniform Detection Assay (FELUDA) by harnessing this specificity of FnCas9 for coronavirus detection during the COVID-19 pandemic. This story is saved for later.” I grinned and continued, “Pasting the correct DNA can only happen once the DNA cutting is done. If the cutting of DNA itself is compromised, how can pasting happen! So, we had to engineer the FnCas9 protein to make it robust for DNA cutting. With this engineered protein we found that the efficient DNA pasting becomes much higher just as we anticipated.”

Neeri interrupts me saying, “That means now, you have a Cas9 enzyme which is very specific and efficient at the same time.”

“Absolutely so! And from there on, we moved to our final goal of correcting genetic diseases. At that time, India had many Sickle Cell Disease (SCD) cases. The production of a faulty protein due to a single letter change in the DNA from A to T can lead to an SCD phenotype. This seemingly simple genetic change made SCD an attractive candidate for CRISPR editing” I say.

“What happens to the patients with this disease?” she asks.

I explain, “Imagine that a water pipe gets clogged by debris which is impeding the smooth flow of water through the pipe. Similarly, the pipe-like structures that we call blood vessels, which are carrying oxygen to every cell of our body, get clogged. This clogging is due to the deformation from natural disc-like cells to sickle shape. These cells are called Red Blood Cells (RBCs) and they flow within the blood vessels.’’

“Are these cells red in colour?” she asks.

I answer, “Yes. These cells carry a protein called haemoglobin which gives our blood its red hue. It is an iron containing protein and by which oxygen gets transported to each of our cells via blood vessels. In SCD, this haemoglobin protein becomes faulty which makes the blood cells sickle and clogs the blood vessels. This causes a condition called anaemia.”

“So interesting!” she exclaims.

I add, “We corrected the A to T misspelling in the DNA with engineered FnCas9 and...” “You cured the disease!!!” she yells in excitement.

My eyes are wide open, looking at my lab mate, utterly perplexed.

“Who’s Neeri!! What are you blabbering?” my lab mate asked.

Now, I realize that I dozed off between experiments and was dreaming in my lab. My dream bubble just got busted and its 2022. I’m juggling with PhD and life. Today I would be setting up the experiment on human hematopoietic stem cells (HSCs) with engineered FnCas9 with the hope to correct SCD mutation in Indian patients.


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