What is CRISPR-Cas 9? Part 1

7 min readSep 7, 2020

Part 1: Mechanism, dangers, and ethical concerns.

When you hear the word CRISPR, what do you think of? A bag of crispy potato chips? That’s definitely what I thought of when I first heard the term. What if I told you that CRISPR-Cas9, a gene-editing system proposed in 2012 by Jennifer Doudna and Emmanuel Charpentier, is a means of editing, deleting, or adding specific sequences to a specific site in your genome? That it has the potential to cure genetic diseases ranging from sickle-cell anemia to muscular dystrophy, and possibly cure cancer? I do not know about you, but I am definitely intrigued. But first, let’s take a step back.

First of all, what is CRISPR?

You asked for it. Clustered regularly interspaced short palindromic repeats. I know- that’s a mouthful. A CRISPR, also known as a CRISPR array, is a type of DNA sequence present in bacteria. When a virus attacks the bacteria, a part of the virus’s DNA sequence is captured and used to make a CRISPR array.

Viruses can have DNA or RNA, but in this example, I am assuming that the virus has DNA. CRISPR arrays consist of units called spacers and repeats. The repeats are sections of repeating code present in the bacteria, and the spacers vary, each being obtained from the genome of viruses that have previously attacked the bacterium.

How do the Bacteria use this?

This CRISPR array allows the bacteria to remember the virus, just like our antibodies remember a specific antigen. When the same virus attacks again, instead of a spacer forming, the viral DNA sequence of that specific virus is transcribed into RNA from the CRISPR array(from the relevant spacer) and is attached to a Cas protein, which comes from a gene which is in close proximity to the CRISPR array.

Note that just one strand of the spacer will be transcribed as RNA is single-stranded and cannot fit both strands of the spacer. For this to happen, the RNA will have to form complements to the opposite strand(template strand). This RNA sequence is attracted to its complementary DNA sequence in the viral genome.

This is how the Cas enzyme can find the virus. After the Cas enzyme arrives at the site, many other Cas proteins are recruited to complete the process. Viral DNA is cut and the virus is disabled. Voila! Now, how does the Cas enzyme differentiate between viral and bacterial DNA?

What if the RNA attaches to a DNA sequence of a bacteria that somehow becomes single-stranded and bacterial DNA gets degraded instead? That would be bad. Well, viral DNA has sequences that are specific to them only. These are known as PAM sequences. So if the Cas protein identifies a PAM sequence in the DNA, it will know that it is viral DNA.

How can that be used for us?

The CRISPR-Cas9 gene-editing system works similarly. Scientists create a short sequence of RNA in the lab with a “guide” sequence that is complementary to a specific DNA sequence in the genome. The RNA binds with Cas9, which cuts the DNA(both strands) at that particular site in the genome. To get these components into a eukaryotic cell, they are loaded onto a vector. Along with the Cas9 and guide RNA, the vector also has the “healthy” version of the “target” DNA.

So, once Cas9 cuts the DNA at that site, the new DNA is added. Scientists make use of the cell’s own DNA repair mechanism to incorporate that new DNA sequence and “seal” the cuts. Usually, a process called non-homologous end joining (NHEJ) or Homologous end joining occurs to repair the double-stranded break(DSB), but it is not accurate and can lead to several mistakes. (It is called “double-stranded” because DNA has 2 strands).In addition to adding a new DNA sequence, CRISPR-Cas9 can also silence or degrade a gene.

Dangers and ethical concerns

Well, if CRISPR-Cas9 can be used for cutting and editing genes, then why isn’t the next race of super-humans being produced- or why doesn’t everybody have blue eyes? As you guessed, it’s not that easy. While CRISPR-Cas9 does have immense potential, there are many things that could go wrong.

1. A series of studies have suggested that CRISPR-Cas9 could do more to damage genes than fix them and may cause cells to lose their cancer-fighting ability.

2. Because of what CRISPR-Cas9 can do and because it is not understood well enough yet, there are restrictions to the process being done on germ-line cells(cells that form the sperm/egg).

Changes made to the germ-line could be inherited by progeny; so, if there is no restriction here, changes can be made that can be used to heighten intelligence, athleticism, and even the appearance of babies.

3. Despite all that scientists know about DNA, there is still so much that is unknown. What if a small base change leads to a change of over a hundred base pairs further down the genome leading to the mutation of several important genes?

4. Also, if a guide RNA, hypothetically, has a sequence ACCCGU and is made to bind with a gene TGGGCA, there are many problems that could arise with this. First of all, the gene in the specific subject wouldn’t look exactly like that, it would be mutated. The goal of this tool, after all, is to attempt to fix mutations in genes that are leading to a certain condition.

So, what if the guide RNA, instead of attaching to the desired gene, attaches to another sequence TGGGCAGG that contains the same sequence and codes for a crucial protein? What do you think would happen?

The protein would likely not be made leading to an important function not being carried out. This is called “Toxic loss of function”. This is just one of the many things that could go wrong. There could be many epigenetic factors too.

5. The ethics behind gene editing is of great debate as of now. CRISPR-Cas9 is not only a scientific issue but also a social issue. Using it to “edit” physical appearance could worsen racism. People would be encouraged to be embarrassed by a certain color of skin or certain sexuality and look to a certain type of people as “ideal”.

In my opinion, a great tool such as CRISPR-Cas9 should not be used for enhancing physical and mental abilities, as this encourages people to adhere to stereotypes of what the “typical” human should be like. This would remove all individuality and strip people of what makes them so special.

In the future, possibly hundreds of years later, when CRISPR becomes safe enough to use, how would a mother feel if her child came up to her and said “Mama please I don’t like my nose, it’s so ugly. Can I change it?” I think the child should be told that she should be happy for what she has and instead consider the girl who has been fighting cancer for 2 years and may now have a fighting chance because of this amazing gene-editing system. Who knows what CRISPR-Cas9 can do in the future? But to ensure its longevity, it should only be employed for people who need it the most.

Thank you for reading this blog post. It means a lot to me. To read part two of this blog series “What is CRISPR-Cas9?”, which will be about experiments and advancements in CRISPR-Cas9, visit https://www.bioffinity.org/. If you have any feedback for this blog or any queries, feel free to contact me. Till then, stay safe and happy.

-Divya Vijay :)

References:

Photo credits: The Scientist Magazine.

1.https://www.geneticsandsociety.org/internal-content/what-human-gene-editing

2.https://ghr.nlm.nih.gov/primer/genomicresearch/genomeediting

3.https://www.sciencedirect.com/science/article/pii/S1044579X17302742

4.https://www.wired.com/story/wired-guide-to-crispr/

5.https://www.nature.com/articles/s41467-019-10366-y

6.https://www.nature.com/articles/d41586-019-00673-1

7.https://www.nature.com/articles/d41586-020-00655-8

8.https://www.the-scientist.com/news-opinion/first-patient-receives-in-vivo-crispr-editing-67222

9.https://www.healthline.com/health-news/crispr-gene-editing-used-for-the-first-time-inside-a-persons-body#What-it-might-mean-in-the-future