CRISPR/Cas9: A Step Forward in the Biological Revolution

Want to know more about CRISPR/Cas-9? This is the article for you!

It’s Sunday afternoon, and you’re watching the news, a bowl of carrots in your hand. Suddenly, the headline reads: BREAKING NEWS: A MUTATION FOUND IN ORANGE CARROTS LINKED TO CAUSING COLOR-BLINDNESS!

You drop your bowl of carrots. The horror! The despair! Everything that makes carrots our favorite orange vegetable — soon to be eradicated forever!

But how in the world are they going to get the orange out of the carrot?

That’s where CRISPR — Cas9 and gene editing comes in. In essence, CRISPR-Cas9 is the copy and paste function on your computer. The crazy thing?

Before CRISPR-Cas9, there was no way of editing an organism’s genetic makeup. The biological process that inspired CRISPR-Cas9 took tens of thousands of years to perfect, but now, we can edit genes with the snip of an enzyme.

What is Gene Editing?

(If you’re someone who already knows basic biology, and just wants to learn more about CRISPR/Cas-9, then feel free to skip this section!)

Gene editing is the process of actually altering DNA. You can add, delete, or alter gene sequences. But what’s DNA?

DNA is the molecule that contains the code for life, and it’s stored in the nucleus of a cell. Everything an organism needs to develop, live, reproduce — you name it, DNA has the instructions to do it! In this case, DNA is what makes a carrot orange. Your genome contains all of the DNA in your body — all of what makes you, you!

DNA has a phosphate and sugar backbone and is made up of four nucleotides (also known as bases): adenine (A), cytosine ( C ), guanine (G), and thymine (T).These bases are arranged in pairs: A — T, and C — G.

A section of DNA displaying A’s, C’s, T’s, and G’s

What if I told you that you have 3 billion of these base pairs in just your genome?

But Apurva, that’s a lot of base pairs in just one genome…what’s keeping them together?

Well, my friend, that would be the double helix: DNA’s shape! The nucleotides are attached to one of two phosphate and sugar backbones and the base pair bonds keep the two strands attached.

So, we’ve talked about how many base pairs there are (3 billion) — that’s a lot of genetic material! It’s stored in the nucleus as a total of 23 chromosomes.

Two X chromosomes — one from your dad, and one from your mom. Taken from UConn Today

DNA contains instructions for making proteins. It goes through a series of processes, like transcription and translation. Unfortunately, these processes often cause a base to be added or deleted and a domino effect occurs:

Base is deleted -> sequence gets read wrong -> protein is completely changed -> things don’t work right in your cells.

Gene editing technology helps us correct these mistakes, so the cells can do their jobs. There are many technologies that can help us do this, but none are nearly as effective as CRISPR — Cas9.

WHAT IS CRISPR-Cas9?

CRISPR stands for Cluster Regulated Interspaced Short Palindromic Repeats. Although the system has many Cas proteins it can work with, the Cas-9 protein gets the spotlight today.

It was inspired by the way bacteria defend themselves against viruses.

Here’s how they do it:

  • The bacteria will cut out pieces of viral DNA (known as protospacers) using the Cas1 or Cas2 proteins and insert it into their own genomes, between the genome’s CRISPRS. The resulting sequence is called the CRISPR array.
  • When the virus comes back, the bacteria is armed and ready! The process starts with crisprRNA (crRNA) transcribing the CRISPRS and the pieces of viral DNA. The crRNA’s are generated by a compound known as the CRISPR locus.
  • Then, tracerRNA (tRNA) links up with the transcripted pieces of DNA through base pairing.
  • Cas9 trims this RNA+DNA compound down so that Cas9 can search for viral DNA more efficiently
  • With all of this, the next time the same virus attacks, the bacteria will recognize it and eliminate it in a snap!

Wait — if the viral DNA and that of the CRISPR array are the same, how does Cas9 know what to look for?

Introducing the Protospace Adjacent Motif, or the PAM! This is a short sequence (2–3 bases long) that follows the protospacer as it’s inserted into the bacteria’s genome. The PAM has to be present for the Cas9 to have the A-OK for cutting the genome.

But how come Cas9 doesn’t cut the bacteria’s own genome? The key is that the CRISPRS within the bacteria’s genome do not match up with the viral PAM. Cas9 directly matches a specific sequence, and each of the bacterial CRISPRS has a slightly different sequence. Thus, Cas9 can only cut that sequence and destroy it, whereas with the bacterial nucleic compound, it will simply bind.

That’s the necessary background info — let’s get to the fun stuff!

The Main Players:

  • Cas-9: a protein that acts as a pair of molecular scissors for cutting DNA.
  • guideRNA (gRNA): this is a predesigned piece of RNA (a nucleic acid like DNA) that matches up with the target DNA sequence. Based off our friend PAM — each Cas protein has a specific PAM it recognizes (and therefore has only certain types of gRNAs it works with)
  • And of course, the sequence (of A’s, T’s, C’s, or G’s) that you want to work with.

How it works:

The process may be different when plant and animal cells — but it works pretty much the same.

CRISPR-Cas9 in action! Taken from flyCRISPR
  • gRNA guides Cas9 to the target DNA sequence. The gRNA should have the RNA bases complementary to the target DNA -> the gRNA will bind to the target sequence, and make a copy.
  • Cas9 will bind with gRNA and the tRNA. The tRNA will loop around on itself to form a “handle” that the Cas9 actually holds onto.
  • Cas9 will cut the target DNA sequence out along with the gRNA.

The cell will be repaired in one of two pathways:

  • Non-homologous End Joining (NHEJ): in which the cell will use proteins to create a DNA pair-end complex, which will join with compatible bases of the cut DNA sequence. Can often cause insertion or deletions of bases.
  • Homology Directed Repair (HDR): DNA template is inserted with gRNA or Cas-9 (or Cas-protein). The DNA template must contain the addition you want as well as a few other homologous bases in order to minimize error.

What can we use CRISPR / Cas-9 for?:

CRISPR / Cas9 ‘s ability has a bunch of applications — these are just some of the few!

  • extracting HIV by removing HIV-1 DNA from T-cell genomes
  • gene silencing by removing an entire sequence out of a gene — by observing what the lack of a certain gene causes, we can learn more about that’s gene function
  • improving IVF by editing the DNA of human embryos
  • creating biofuel through editing the DNA of algae

What’s being done in the lab right now?

Recently (and we’re talking just a couple days ago, at the time this was published) lab researchers have done the following:

These are just a few of the things CRISPR/Cas-9 can do!

The ethics:

Let’s get back to our soon-to-be-not-orange carrots. We know that removing the sequence that causes a carrot to be orange will potentially prevent color blindness. But what if not having that sequence gives rise to another disease, like going fully blind.

Seems counterintuitive — you’re causing harm when you were trying to take out something harmful in the first place!

Because scientists are not sure what every single gene sequence in the human genome encodes for, it’s hard to know for sure what removing one will do. That’s why countless experiments have to be done in the lab before it can safely be used on humans in the future. (looking at you He Jiankui).

Speaking of designer babies, the next generation of parents will have the choice to edit genes that will give their kids better hair or a certain color of eyes. It seemed like science fiction in the past, but it’s becoming a very real possibility. (again, hello He Jiankui!)

On one hand, future generations could become superhumans, with extremely advanced muscles or eyesight. We’d reduce the amount of diseases we can get, eliminating things like cancer or dementia.

The future Average Joe’s arms could look like this, thanks to gene editing.

On the other hand, there are certain socio-economic gaps that could very well increase. Those in third-world countries, who wouldn’t have much access to gene editing tech, would be drastically different from the superhumans of first world countries.

And then, you have people that will misuse use the wonders of gene editing tech for their means. Do we have a mad scientist working on a private army of supersoldiers already?

It all sounds scary, I know. But the more people that are educated on tech like this, the more people can use it responsibly — they’ll know how to optimize the use of CRISPR/Cas-9 and similar technologies so ultimately, everyone can benefit!

People will be able to see how much CRISPR/Cas-9 holds for the future — and how it’s the first step in the biological revolution.

TL;DR:

  • Gene editing is the process of altering DNA, which contains the instructions for life.
  • CRISPR-Cas9 is just one of many gene editing tools that works to cut DNA sequences out; it can repair and add gene sequences back in.
  • The system is based on the way bacteria defend themselves against virus.
  • Cas9 is the protein that does the cutting, and guideRNA makes sure Cas-9 is removing the right gene sequence.
  • Gene editing tech can be used for good and for harm (from editing things like eye color to creating an army of super soldiers)
  • The biggest thing we can do is educate the masses about this exponential tech, and others like it, so it can be used responsibly.

Thanks for reading this article! I hope you were able to learn more about CRISPR-Cas9 and how awesome it is! If you’d like to talk more about gene editing with me, this is my Linkedin, and you can email me at writetoapurva@gmail.com. Until next time!

Hey there, my name’s Apurva! I’m passionate about the applications of genetic engineering, and currently I’m working on human limb regeneration!