An Intro to Gene Editing- CRISPR Cas9
By: Nikhita Srinivas
Tags: Biology, CRISPR, Gene Editing, Science

Would you want to design a human? Edit its embryonic form to change its physical appearance, immunities, and intelligence levels before it is even born? Create your ideal human. That sounds crazy. It sounds like science- fiction! However, the technology for this already exists.

Gene editing allows us to make changes to our fundamental genetic code, DNA. Early gene editing technology was invented in the late 1900s and when Jennifer Doudna invented CRISPR Cas-9, it became easier than ever.

The Basics:

As humans, we are made of cells. Each cell has a copy of our genome, all our genes and DNA. Humans have 20,000 to 23,000 genes. Genes consist of DNA which is the code or “recipe book” that is used to create proteins (large, complex molecules that are responsible for the structure, and function of organs and tissue in our bodies).

DNA molecules are in the shape of long double helixes (these are similar to spiral staircases with millions of steps). Each step on the staircase is a different type of molecule called nucleotide bases. There are 4 different types, Adenine (A) which is always paired with Thymine (T) and Guanine (G) which is always paired with Cytosine (C).

Different combinations of 3 nucleotide bases result in the creation of different proteins. For example, GCT will not make the same protein as GTT.

The process to turn the coded genetic information to a protein involves transcription and translation. However, these processes can mess up and a nucleotide base can be added or deleted. When this happens, the DNA bases change, the subsequent protein is not the right one, and your body does not function the way it is supposed to.

DNA breaks a lot. However, it is normally uneventful since our cells have built- in DNA repair processes that constantly fix those breaks as they occur. However, if breaks are not addressed properly, mutations can occur. This can lead to serious diseases such as sickle cell disease or cancer.

Can we solve these problems?

Introduce CRISPR Cas9- a gene editing tool we use to modify, delete, or correct precise regions of our DNA.

How does it work?

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats

It is based on a system that bacteria use to protect themselves from viruses.

How does that system work?

When a virus invades a bacterium, the protein complex known as Cas1 and Cas2 identifies the viral DNA and cuts out a segment (this is known as a protospacer). The protospacer is inserted into the front of a CRISPR array. This is a short stretch of DNA in bacteria that is composed of alternating and repeating sequences called spacers. These spacers collect the DNA of invading viruses. When the virus returns, the bacterium has a memory of the infection and can defend itself. The bacterium starts by transcribing a CRISPR RNA (crRNA) from the spacers in the CRISPR array. A separate RNA (tracrRNA) connects with the crRNA The Cas9 protein trims the RNA strands so they can search for the viral DNA If the protein complex finds a sequence in the invading virus that matches the sequence in the crRNA, it will cut the viral DNA and disable the virus!

But since the DNA in the CRISPR array is the same as the DNA in the virus, how does the Cas9 protein know the difference?

PAM is a short sequence of nucleotides, around 2–6 base pairs, that comes after the protospacer sequence in a viral genome. The spacer sequence in the CRISPR array is not followed by a PAM sequence. The PAM sequence MUST be there for the Cas9 protein to actually cut the DNA.

Back to gene editing!

CRISPR Cas9 technology consists of two parts: the Cas9 enzyme and a piece of RNA called a guide RNA (gRNA)

The Cas9 acts as a pair of “scissors” that can cut the DNA at specific locations in the genome so DNA can be added or removed.

acts as a pair of “scissors” that can cut the DNA at specific locations in the genome so DNA can be added or removed. The gRNA consists of an RNA sequence that is around 20 bases long. This located in a larger RNA scaffold. The guide RNA recognizes the target RNA region and directs the Cas9 enzyme there for editing.

This is how CRISPR Cas9 Works:

Scientists create a guide RNA that is complementary to the specific sequence they want to edit. The guide RNA finds and binds to that specific DNA sequence. The Cas9 enzyme goes to the same location as the guide RNA and cuts both strands of DNA

After this, 1 of 2 things can happen

Non- homologous End Joining : The cell tries to fix the cut by connecting the ends without a template. This is error prone and normally results in mistakes which can lead to random mutations that disable the gene. This can also cause an insertion or deletion of bases.

: The cell tries to fix the cut by connecting the ends without a template. This is and normally results in mistakes which can lead to random mutations that disable the gene. This can also cause an insertion or deletion of bases. Homology Directed Repair: When researchers need to be more precise (replacing a mutant gene with a healthy copy), they add another piece of DNA to the CRISPR complex that is the desired sequence. Once the cut is made, the cell’s repair system will pair the DNA template with the cut ends and replace the original sequence.

Applications for CRISPR Cas9:

CRISPR has many applications! These are only a few…

Therapy for blood disorders- CRISPR Therapeutics and Vertex Pharmaceuticals harvested bone marrow stem cells from patients and used CRISPR to produce fetal hemoglobin. Gene silencing- by silencing a gene, scientists can learn about their functions Genetically modifying food- We could create foods that resist bacteria and viruses or even just create better tasting food. This is already being done! Genetically modifying human embryos- this technology could be used in the future to create “designer babies”

What are the Ethics?

Gene editing comes with many ethical questions.

What should we use CRISPR for and, possibly more importantly, what shouldn’t we use it for?

For people who have been suffering from genetic diseases, CRISPR has the potential to be life changing. It is the possibility of curing major medical problems such as cancer and Alzheimer's. However, going back to the idea of designer babies, is that a good idea? Is it ethical to allow parents to edit their children’s eye color, intelligence, and athleticism? It could prove to be beneficial. We could significantly reduce the risk of diseases such as cancer or cystic fibrosis and create children with enhanced capabilities. However, we would also increase the socio- economic gap between third and first world countries. Poorer countries would not have the access and money to use this technology and would not gain the same benefits as those in first world countries.

Then there are also safety concerns. What if CRISPR could cure sickle cell anemia but in the process, removed the cell’s ability to fight cancer? If in the process of curing something harmful, it introduced something potentially more harmful. There is so much we still do not know about how our genes work and editing them could lead to unknown repercussions.

However, as scientists research further into CRISPR, they learn more about the risks, rewards, and limitations, and more people are able to benefit from this life changing technology!