An Exploration of Gene Editing: CRISPR/Cas9

You’ve probably been told sometime in your life that you look like your parents. Whether it was that aunt you’ve never even met telling you how much your nose resembles your dad or that old lady next door that talks about how much your eyes look like your mom’s, you’ve experienced it at least once in your life.

That was most likely the result of your genetics. After all, you got all of your genetic information passed down to you from your parents. So, you are bound to have a lot of resemblance to your family.

When this concerns your resemblance to your parents, it’s not that big a deal. But when it comes down to things like inheriting genetic disorders, it can be very crucial.

Today, most people with inherited genetic disorders have no choice but to suffer from their disorders for life. There may be treatments that ease or attempt to ease the suffering, but there is nothing that can completely cure the disorder, and make you better.

Until recently.

With the discovery of gene editing, especially with CRISPR, this idea of an incurable genetic disorder is quickly being ripped apart piece by piece, until it soon, it will have gone from an incurable genetic disorder to just a temporary inconvenience.

So let’s explore the world of gene editing, by analyzing the most hyped method for the process — CRISPR-Cas9


CRISPR was definitely not the first method of editing our genome. So why all the hype?

Because all the ways utilized before were expensive, time-consuming, and inaccurate.

Why was CRISPR better? First, it was relatively cheaper to produce and did not take as much time or effort to design. Second, it had much higher accuracy than all the previous methods, making it possible to effectively and efficiently edit the genome to achieve the desired purposes.

So What Is It

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It was first identified in prokaryotes in 1993 as a set of palindromic segments (same front and back) of DNA that were interspaced with other genetic material.

In 2007, researchers finally concluded that CRISPR functioned as the prokaryotic immune system.

See, when we think of the immune system, we often only think of the system in human and animal bodies, aimed to protect us against bacterial and viral infections.

But prokaryotes like bacteria are not too different. They have to protect themselves too. From what?

Viruses. Every day, billions of viruses around the world are constantly attacking and killing off billions of bacteria. This means that the prokaryotes have developed ways to fight against the viruses, one of which is the CRISPR system.

Viruses that attack bacteria are also known as bacteriophages

CRISPR acts as the immune system against viruses that undergo the lysogenic cycle, where, instead of immediately reproducing and killing the host cell, the virus incorporates its DNA into the host DNA. Later on, when environmental conditions are favourable, the virus is triggered to begin reproducing and finally kills off the host cell. CRISPR’s job is to find these foreign viral DNA sequences, and, using Cas proteins, snip them out.

In addition to this, much like the human immune system, the CRISPR system also “memorizes” this specific sequence by storing a spacer sequence (a piece of the viral DNA) into the palindromic sequences of the CRISPR array. So, when a virus with the same sequence attacks again, CRISPR is ready to find the DNA and cut it out.

CRISPR Structure

The CRISPR system consists of two components required to target specific genome sequences and make a cut in the DNA.

The first component is the gRNA (guide RNA) molecule. This molecule plays the role of guiding the CRISPR system to the targeted genome sequence. The gRNA molecule itself also consists of two distinct RNA segments (it’s like Matryoshka dolls; open one and you get another): CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA). crRNA is an 18–20 base pair sequence that binds to the genomic target, and tracrRNA acts as a scaffold for crRNA and Cas interaction. In their natural form, the crRNA and tracrRNA anneal together to form the gRNA.

The second component is the CRISPR-associated endonuclease (Cas), which functions as the molecular scissors that cut the genome at the targeted site. The most common form of the Cas protein utilized in research applications today is Cas9.

How It Works

Once the CRISPR system has entered the cell/nucleus, it keeps bumping into the genome attaching to sites known as protospacer adjacent motifs (PAMs), differing for each Cas molecule used. For the Cas9 protein, the PAM sequence is 5'-NGG-3', where N is any nucleotide. Every time it attaches to one of these sites, it unzips the DNA beside the PAM slightly and compares the crRNA molecule to the DNA. If there is no match, it continues on, one PAM site to another. Once it does find a site where the genome is exactly the same as the target sequence, however, it can get to work.

Once the target sequence has been found, the Cas9 nuclease forms a double-stranded break at the location, knocking the gene out of commission. If the purpose of the CRISPR was to turn the gene off, this is enough. The cell’s natural repair mechanism, known as non-homologous end joining (NHEJ), will take over after and insert or delete nucleotides (called indels) to connect the severed strands. This induces frameshift mutations that change the protein produced, to remove the impact of the target gene.

However, this method, also known as knockout gene therapy, designed to turn off a gene, can be highly risky. The changes the cell will make are entirely random, and may even result in a new sequence of nucleotides that has much more harmful effects.

In many cases, scientists and biologists instead use knock-in gene therapy. This process replaces the malfunctioning or mutated gene with a functioning/desired gene using homology-directed repair (HDR). To carry out this process, a DNA donor template with the desired replacement sequence, flanked by homologous regions, must be introduced with CRISPR. This sequence is used to repair a broken sequence via homologous recombination (exchange of genetic material between similar DNA strands), to incorporate the desired changes.

Different CRISPR Applications and Methods

CRISPR Interference (CRISPRi)

For this procedure, a dead Cas9 (dCas9) molecule that doesn’t cut the DNA is used. Instead of cutting, the dCas9 binds to the target sequence and prevents transcription of the gene.

This is achieved by fusing a transcriptional repressor domain to the dCas9 molecule, which would cause a reversible and accurate reduction in the expression of that gene.

CRISPR Activation (CRISPRa)

This is a lot like the CRISPRi mechanism, except the dCas9 molecule is instead used to activate the genes. Scientists can fuse transcriptional activator domains to overexpress the gene of interest, increasing the amount of protein coded by that gene.

CRISPR Screens

Surprisingly, despite how long we have been working with DNA, the function of much of the coding regions in the genome is still unknown. The main problem is the inability to directly connect a specific genetic sequence to a specific protein and function in the cell.

The CRISPR screening process is carried out to determine the purpose of specific genes at a genome-wide level. By doing this, researchers can obtain information like the causes behind certain disorders, identify targets for drugs, and just gain a deeper understanding of how the cells function.

To carry this out, researchers can use either knock-out therapy, CRISPRi, or CRISPRa. By inactivating or over-activating the specific genes, researchers can determine the effects of the changes on the cell, including changes in the protein levels, allowing them to determine what each gene does.

Gene Visualization

What if, instead of determining what gene does what, you only want to know where a specific gene or genome sequence is located? This can be achieved with gene visualization.

To achieve this, fluorescent proteins are attached to the dCas9 molecule, which then bonds to the target sequence. When the fluorescent proteins emit light, it can be detected by computers to determine the location of the gene.


Multiple gRNA molecules targeting the same gene can be used together to have a much larger impact. This can be used especially for knock-out gene therapy to induce large fragment deletions, such as the deletion of entire genes.

The Problem with Adult Gene Therapy

But, most of the disorders CRISPR aims to treat, such as cancer, only properly develop once the individual has grown. And by this point, it might be too late.

See, CRISPR can edit the genes in one cell. When the disorder is based on a small population of cells, such as a small cancerous tumour, this is not that big a deal, since we can just use CRISPR in all the cells. But when you have genetic disorders like cystic fibrosis or Huntington’s disease, the population of cells affected is much much larger. And to edit all those cells, it would require lots of time, effort, and money to prepare a large amount of gRNA and Cas9 molecules. This makes editing genes in adults to cure disorders very inefficient and expensive.

This is why researchers are also focusing on developing the ability to predict the development of disorders before the zygote development goes past the blastula stage. By finding a problem in this phase, researchers can use the CRISPR system to edit the small population of cells, which can then reproduce into an individual made of healthier, cured cells with the edited DNA.

The Sad Truth

So, the truth is, someone who’s inherited a genetic disorder from their parents might have to wait for gene-editing technology to reach a stage where adult cells can be edited on a large scale before they can be cured.

But that doesn’t mean all hope is lost. Researchers are continuously making strides in gene-editing technology, making the process better on a regular basis. So, even though adult gene therapy needs more time, it’s definitely not far in the distance. Who knows? It might even be developed in the coming decade.

Hey guys! This is Akshaj, a 16-year old innovator obsessed with artificial intelligence, gene editing, and biomedical technology. If you liked the article and learned something new, please leave some claps to show your appreciation. Also, if you want to read more from me, follow me on Medium.

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That’s it for me. Signing off for now. Until next time.



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