The Process Of CRISPIR Explained Simply in X minutes
For the past month I have been exploring the endless possibilities of world changing technologies that I could focus on. During my expedition I studied technologies like: Brain Computer Interfaces, Biological Computing, Artificial Intelligence, and even Fusion energy. However, in all my exploring the one technology that I feel as if I could learn about for hours upon hours is Gene Editing. Gene Editing is quite self explanatory, its definition on the internet is: the process that lets scientist edit the genes of multiple organisms. Like every world changing technology, its applications are limitless. From producing fruits and vegitables that are resistant to pesticides and insects, to editing the next generation of superhumans, Gene Editing’s capabilities are plentiful. Nevertheless, Gene Editing isn’t what we will be discussing today. Have you ever wodered how we are able to edit our genes successfully? Well, thanks to a process called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Gene editing is made possible. So, what is CRISPR? To understand CRISPR we must first understand what it is used to edit: DNA.
DNA is short for Deoxyribonucleic acid which is what determines every single attribute in our lives. From the colour of our eyes, to whether or not we prefer to read rather than play video games, DNA is what makes you unique. Take a look at the picture to the left, what you see is a piece of DNA. Some initial observations that we can make is that DNA takes a double helix shape, and DNA appears to be made out of 5 different components. The reason that DNA takes its double helix shape in order to prevent the 4 nitrogenous bases (Adenine, Thymine, Cytosine, or Guanine) from having contact with the cell fluid. For our second observation, don’t let initial observations fool you. Let’s take a look at DNA on the molecular level.
The Image you see above is a single strand of DNA nucleotide. As you can see, a single molecule of DNA is made up of 1 phosphate group, deoxyribose, and one of 4 nitrogenous basses.
Phosphate group
The phosphate group forms the famous double helix structure of DNA, which consists of two linear strands of sugar-phosphate backbones that run over one and otehr to form a double helix shape. The phosphate group is also negatively charged; this aspect of the phosphate group is extremely important since it makes DNA as a whole negatively charged so that it could repel neucliophiles, which limits its risk of neucliophilic attack.
Deoxyribose
The next part of the DNA nucleotide is deoxyribose, which acts as the “middle man” for DNA. Its job is to attach to the phosphate group and carry the nitrogenous base. Due to its molecular structure, the deoxyribose forms a strong phosphodiester bond with the phosphate group. phosphodiester bonds form when two hydroxyl groups in phosphiric acid react with the hydroxyl group on other molecules forming ester bonds. To learn more about phosphodiester bonds check out this site. Deoxyribose is also able to make strong bonds with the nirtogenous basses through glycosidic bonds. Lets take a look at a diagram to better understand:
Firstly, let us talk about the ester bond that is formed between the phosphate backbone and the deoxyribose. As highlited by the smaller red circle, the hydrogen in the phosphate group reacts with the HO in the deoxyribose to form water. The water is removed, which creates an ester bond between the Carbon molecule and the oxygen from the phosphate group.
Secondly, let us talke about the glycosidic bond that is formed between deoxyribose and adenine in this diagram. The hydrogen atome of adenine reacts withe the hydroxyl in the deoxyribose to create water. the water is removed and a bond betweent th carbon molecule bonds with the nitrogen in the adenine creating a glydosidic bond. However, the bonding between the C and the N only happens with purines such as Guanine and Adenine. So, what happens when Cytosine or Thymine bond with deoxyribose?
In this case, when thymine binds with the deoxyribose the Hydrogen atome reacts with the hydroxyl once again to create water. The water is removed and a bond between C1' and N1' is formed.
Nitrogenous base
Finally, let us talk about the most important part to DNA: Nitrogenous bases. There are 4 different types of nitrogenous bases:
and depending on how the bases are sequenced, our body could have a higher risk of type 2 diabetes than someone with a different sequence, or we could have blue eyes instead of green. The nitrogenous bases is our body’s way of storing information. So, what exactly are the bases?
Two categories of Nitrogenous bases
The first category of Nitrogenous bases are the purines, Adenine and Guanine. The purines are composed of a double ring structure (as seen in the pictures above) which is the only visible difference between pyrimidines: Thymine and cytosine. Pyrimidines only have one ring structure.
Base pair bonding
One major rule when it comes to DNA is that Adenine will always bond with thymine, and Cytosine will always bond with Guanine; this makes sense for two reasons: the double ring structure of purines bonding with the single ring structure of pyrimidines helps keep the DNA size consistent, and because their bondage is chemically favorable. Lets take a look at a diagram for some further clarification.
As you can see, bases are connected through hydrogen bonds. the oxygen,and hydrogens of guanine make exactly three bonds with the hydrogen, nitrogen and oxygen of cytosine. However, the hydrogen and nitrogen of adenine only form two bonds with the hydrogen and oxygen of thymine; this is why A and T, and C and G are perfect for eachother, each base has enough bonds to stabilize the other. To learn more about nitrogenous bases, check out this really cool video I found:
So, what is CRISPR?
Now that we’ve studied DNA a bit more, lets learn the process of editing it. CRISPR, or Clustered Regualarly Interspaced Short Pandromic repeats, is the natural process of editing DNA. In nature, CRISPR is used as part of the bacterial immune systems to set up a significant defence against any invading virus. CRISPR essentially edits the bacteria’s DNA to be immune to that certain virus.
The process
CRISPR is actually quite simple. For the Gene Editing to take effect the bactirum would need two pieces: short pieces of repetitive DNA sequences (CRISPR) and cas protien. The cas (CIRSPR associated) protien is acts basically like a molecular sicssor that cuts DNA. The first step of editing immunity to viruses in bacteria is letting the virus enter the bacteria in the first place. Once the virus enters the bacteria, the CAS protein cuts a piece of the virus’ DNA out and stiches it to the CRISPR. After the viral DNA is attached to the CRISPR region of the bacteria, the viral DNA is copied into short pieces of guide RNA. The Guide RNA would then attatch itself to a CAS 9 protien, which acts like a weapon that uses the gRNA to lock onto its target and eliminate it. Think of gRNA as facial recognition, it searches the bacteria for a DNA sequence that matches that of the viral DNA and the cas 9 detroys it. Here is a really great video that explains CRISPR really well:
Summary of the video:
- CRISPR was first identified in Ecoli.
- CRISPR stands for clustered regularly interspaced short palindromic repeats.
- the first part of CRISPR are the short palindromic repeats, these repeats of DNA will be around 20 to 40 letters in length and they’re gonna be palindromes.
- “palindromes are a sequence of letters that read the same from left to right” they are never odd nor even.
- the repeats are interspaced, which means there is spaces between these pieces of DNA.
- Between the spaces of the DNA you’ll find spacer DNA.
- Spacer DNA is not identical, each piece of spacer DNA is unique in its own way.
- CRISPR also has CAS genes that are able to create CAS 9 protiens.
- CAS proteins are helocases (protien that unwinds DNA), and neucliases (protein that cuts DNA).
- The initial action that causes CRISPR system to activate is when the virus injects is DNA Into the cell. (without an immune system, the viral DNA will hijack the cell and eventually kill it.) If the viral DNA matches one of the DNA strands in the spacers, the CAS protiens will transcribe the matching DNA into guide RNA which will help the CAS 9 protein kill the virus. If the viral DNA does not match any of the DNA strands in the spacers, CAS proteins break apart the viral DNA and copy it into the CRISPR system. When the virus attacks once again, the CRISPR system will be ready since its added the viral DNA to its data bank.
- Scientists wanted to “highjack” this system to use it to edit DNA. The most popular way to edit DNA with CRISPR is the CAS 9 system.
- A CAS 9 protein is made up of a nueclease (the part that helps with cutting of DNA) and TRACRRNA (bonds with the crRNA to make guide rna the cas 9). Scientist believed if they could change the guide RNA into a sequence of DNA that they wanted to eliminate, and if they could make the tracr RNA attach to the new guide RNA they would have a simple gene editing system. Essentially, the scientist would inspect the DNA for segments they want to edit, create a replica RNA and attach it to the CAS 9. The CAS 9 would then locate the sequence and destroy it. After the sequence of DNA has been cut, you could replace the lost DNA sequence with a new host sequence that can be whatever you’d like.
Future applications of CRISPR gene editing:
- Can possibly cure a lot of diseases such as blindness (colour blindness and blindness), muscular atrophy and osteoporosis.
- Can edit crops to become resistant to pesticides and droughts
- can possibly edit live stock to produce less waste and require less water
- can edit humans to become more intelligent or have certain physical attributes
TL;DR
- DNA is made up of tiny subunits called nucleotides
- 4 nitrogenous basses
- CRISPR stands for clustered regularly interspaced short palindromic repeats
- CRISPR works by using the cas 9 protein in bacteria to slice certain sequences of DNA using a modified guide RNA and host RNA.
