Many words can be used to describe the CRISPR-Cas9 system, among which I would include innovative, empowering, phenomenal and perhaps even could go so far as to say life changing… well more literally, DNA changing. Yet, that DNA modification cascades and before you know it, that one DNA alteration is life changing.

In fact, just the process of gene editing is incredible, since our genes hold our characteristics and ability to function. While there are various methods of gene editing, one, in particular, stands out. That one technique is CRISPR-Cas9 editing, a unique technology that was developed in 2013.

What led to developing CRISPR-Cas9?

CRISPR/Cas9 inspired genetic editing is a compilation of many researchers and their work including:

• The discovery of CRISPR by Francisco Mojica in Spain in 1993 and his work with it up until 2005.
• Alexander Bolotin, who discovered Cas9 and PAM in 2005 while working with bacteria in France.
• Work with adaptive immunity and how the bacteria S. thermophilus responds to a phage attack done by Philippe Horvath and scientists in France in 2007.
• John van der Oost and his colleagues in the Netherlands in 2008, who showed that in E.coli, spacer sequences derived from the phage are transcribed into CRISPR RNAs that will then guide the Cas proteins to the target DNA.
• The demonstration by Sylvain Moineau in Canada, that proved Cas9 was the only protein involved in the double-stranded break in the target DNA.
• In 2013, Feng Zhang, who had previously worked with other genetic editing systems like TALENs, was the first to effectively adapt CRISPR-Cas9 for editing in eukaryotic cells. He engineered two Cas9 enzymes and performed targeted genome cleavage in human and mouse cells. They showed how the system could be wired to target multiple locations, and how it could be used to drive modified repair.

So what is CRISPR-Cas9?

Simply put, CRISPR-Cas9 is a feat of biomedical engineering that can edit our human genome by removing, adding, or altering sections of the DNA sequence. More specifically it is known as The Clustered Regularly Interspaced Short Palindromic Repeats Type ll system that is used in bacterial immune systems and has been modified for genome engineering.

The CRISPR sequences were originally discovered in E.Coli in 1987, and the knowledge of the system as a coping mechanism against bacteriophages was not realized until 2007. Experiments included exposing S. thermophilus to phages to test if the viral DNA would be incorporated into the bacteria's genome and cause the Cas genes, which coded for polymerases, nucleases, and helicases to be disrupted.

The results were that prokaryotes developed an immune system that used Cas genes to record and destroy the invading phages. The Cas proteins would snip the foreign DNA into small fragments and paste them into the CRISPR sequence, and more Cas proteins would process the CRISPR location and generate RNA, which would guide Cas nuclease to the specific DNA.

Once the complex binds to the foreign DNA, a cut is made to destroy the invader. Since 83% of archaeal and 45% of bacterial genome successfully utilize the CRISPR system, I guess we wanted to use it as well.

Why do we want to use genetic editing and CRISPR-Cas9 technologies?

It always seems that the more complex a process is, the more room there is for error. And no matter how incredibly accurate for how complex they are, there is always room for error in our body’s biological processes, including DNA replication or chromosomal division in mitosis.

Errors such as deletion, duplication, translocation, nondisjunction, etc. cause mutations in DNA which can lead to disorders and diseases. DNA editing techniques are the ways we can fix unwanted mutations, and of the various methods, CRISPR-Cas9 seems to be a quicker, cheaper, and more versatile way of altering DNA.

Why is CRISPR-Cas9 special?

This technique is so efficient and so effective because, well… it's very simple, and it's based on a natural biological adaptation for correcting genes in prokaryotic cells. It was discovered that bacteria had a successful gene editing process that they used to respond to invading pathogens.

The bacteria would snip out parts of the virus DNA to stop infection. Scientists saw the potential and tweaked and mimicked the process so that it could be used in eukaryotic cells.

How does CRISPR-Cas9 work?

The CRISPR-cas9 system has only two key factors behind how it introduces change in DNA.

One of the system’s secret weapons is an enzyme known as Cas9. Cas9 is the analogous pair of scissors that will cut two strands of DNA at a specific location in the genome, enabling DNA to be added, removed or altered.

Now before you assume that the Cas9 scissors are the most important part, remember that scissors aren't effective if they don't cut anything-- or especially if they cut the wrong stuff. This leads to the job of a very important strand of RNA called guide RNA (gRNA), the second key component in the CRISPR-Cas9 system.

This gRNA consists of a very important, small piece of pre-engineered RNA sequence that is roughly 20 bases long and is located within a longer RNA scaffold. The scaffold will bind to the DNA, targeting the DNA sequence that the gRNA was designed to be complementary of. The pre-designed sequence will now guide Cas9 to the right part of the genome, ensuring that it will cut at the right location.

So overall, the gRNA is injected and the Cas9 enzyme follows the gRNA to a location in the genome where it cuts across both strands of DNA, triggering the cell which will recognize that the DNA is damaged and try to repair it.

What’s wrong with the system?

Well, the guide RNA is designed to bind to a specific sequence in the DNA because it has bases that are complementary to those of the target DNA. But this doesn't necessarily mean accuracy because it's not a given fact that cytosine must bind to guanine or that uracil must bind to adenine, and vice versa.

Rather, it's just a very supported, high probability heuristic that is not often, but still open to discrepancies. So in theory, the gRNA will bind to the target DNA, or it could bind to a very similar region in the genome.

A strand of DNA could be fairly close to the 20 complementary sequence leaving potential for the guide RNA to bind there instead. Thus researchers have another task ahead of them: a search to try and eliminate “off target effects” where the system cuts at a different gene than the one that was intended to be edited.

Another issue is about using gene editing in germline cells, which is actually illegal in the UK and most other countries. Currently, CRISPR-Cas9 is only being used on somatic cells, and while it has been used in a small number of exceptional cases to treat humans, research is still focusing on using CRISPR-Cas9 in animal models or isolated human cells.

What does the future hold for the CRISPR-Cas9 system?

Utilizing CRISPR-Cas9 gene editing is a possibility for treating multiple medical conditions that have a genetic component, such as developing treatment for cancer, hepatitis B, high cholesterol, or various other possibilities.

What have we accomplished thus far with it’s application?

• Researchers have used the CRISPR-Cas9 to remove certain DNA in mosquitoes, and when the mosquitos cellular system tried to repair the genome, they manipulated it so the missing DNA was replaced with a DNA construct engineered by scientists. Here's the amazing part-- two genes in the construct make the mosquito immune to the parasite Plasmodium falciparum, which causes malaria. Now I know what you’re thinking: “Sure, but that's just one mosquito." But, the experiment showed that the immune mosquitoes pass on the resistant genes to 99% of their offspring, even when mated with normal mosquitos. So how is that for progress in fighting malaria?
• How about using gene editing to eliminate cancer? Okay, I admit this case didn't involve CRISPR-Cas9, but CRISPR-Cas9 can still be used. This case involved another gene editing process called TALEN. An infant girl with a form of leukemia blood cancer had been undergoing chemotherapy and bone marrow transplants as treatment, however they were not proving to be successful. Doctors decided to use TALEN gene editing technologies in a hail Mary effort to save the girl. They altered T-cells of a donor to more effectively locate and kill leukemia cells without attacking the body, and through this process the doctor successfully eliminated her cancer.
• What about Duchenne muscular dystrophy, that has fewer than 200,000 occurrences per year, but lasts for years or could be lifelong. It’s a disease caused by a mutation on one specific gene that prevents the body from producing dystrophin protein, causing progressive muscle weakness. Well, in January 2016, researchers successfully treated muscular dystrophy in lab mice using CRISPR-Cas9 to cut and repair the gene. Now the question remains of whether it can be used in humans.
• Another application of CRISPR-Cas9 is that of George Church, who lead a team at Harvard medical school using CRISPR to edit 62 genes in pig cells… at once. Ultimately, the hope is that the editing could make pig organs suitable for transplant to humans.
• There is no known way to make the HIV virus permanently inactive, at least not yet. However, researchers believe they can use CRISPR-Cas9 to remove the viral DNA from the patient’s genome. The only problem is that it’s hard to locate HIV DNA in latent cells that don’t demonstrate any of the HIV symptoms.
• In 2016, startup company CRISPR Therapeutics announced a 300 million dollar joint venture to hopefully develop CRISPR based drugs in order to treat heart disease, blood disorders, and blindness.
• Although not in humans yet, researchers have effectively removed a mutation in mice that causes retinitis pigmentosa, a disease that ultimately leads to blindness. After one application of the CRISPR-Cas9 system, the experimental rats could see better than the control rats. In fact, Editas Medicine is planning to use CRISPR to treat the eye disorder leber’s congenital amaurosis by the end of 2017.
• On October 28th of 2016, a team at Sichuan University in Chengdu delivered CRISPR-Cas9 modified cells into a patient with aggressive lung cancer. The researchers removed immune cells from the patient’s blood, and using CRISPR-Cas9, disabled the gene which codes for protein PD-1 (the protein that will normally initiate a stop to a cell’s immune response.) They then cultured the edited cells, increasing their number, and injected them back into the patient. Without PD-1, the hope is that the immune system will not be stopped, and the edited cells will attack and defeat the cancer.
• Chinese scientists have used CRISPR-Cas9 to delete genes that inhibit muscle and hair growth in goats. Without the inhibitors, muscle and hair growth is promoted, leading to more wool and more meat and supplementing the country’s commercial meat and wool industries.
• CRISPR-Cas9 can be used to improve crop disease resistance and environmental stress tolerance. Editing grapes to resist downy mildew, or modifying grass to resist dollar spot disease so the grass is able to combat fungicides are some possibilities for use.
• You know those teacup pigs? Using the gene editing tool TALEN, Chinese researchers disabled the growth hormone in Bama pigs’ fetal cells, creating the small variety of Bama pigs that are sold as pets.

CRISPR-Cas9 and gene editing technologies have a huge future ahead of them, and a big ethical discussion on how much we should be allowed to edit. Nevertheless, gene editing and CRISPR-Cas9 are changing the world, one strand of DNA at a time.

Sources:

https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/crispr-timeline

https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology

http://www.yourgenome.org/facts/what-is-crispr-cas9

https://www.addgene.org/crispr/guide/