Originally posted on The System Scientist.

Recently there has been a lot of buzz about a gene editing tool, CRISPR Cas. For good reason though: it allows a for relatively simple method of deleting and inserting DNA nucleotide base pairs which has enormous implications in curing genetic diseases and preventing the spread of viruses.

How It Works

This CRISPR/Cas system is a prokaryotic immune system that is not unlike our adaptive immune system. When bacteria are infected by a virus, the organism keeps track of the foreign agent by making a strand of nucleotide bases complementary to the viral DNA. This ensures that if/when it’s infected again, the bacteria can identify the virus and destroy it. It is helpful to imagine the virus as a home invader, CRISPR as the criminal’s mugshot, and Cas as the police force who terminate the hostile situation.

Bacteria store the ‘mugshots’ in their DNA by making repeated sequences of genetic code with “spacers” of the viral invader DNA resulting in an archive of past infections. (Technically, these spacers are called Clustered Regularly Interspaced Short Palindromic Repeats, which is where the term CRISPR comes from.) This discovery was one of the first in the development of understanding CRISPR/Cas.

However, rather than a double stranded DNA strand, it’s the corresponding single stranded RNA that is used to identify viral perpetrators. The “mugshot,” needs to be single stranded so it can unwind the double stranded viral DNA and detect its ‘mirror image.’ Normally, it is very difficult to rip apart the helix structure, so this system uses a Cas (CRISPR ASsociated) protein to help find the right DNA sequence.

Another role of Cas within the bacteria is to cut the viral DNA once it’s been identified hence eliminating the threat of infection. It is this function of the system that is being used for gene editing because molecular biologists have realized that any DNA sequence can be identified and cut. From there, either gene knock-out (seeing what happens when a gene is not coded) or knock-in (seeing what happens when a modification is made to a gene) studies can be performed the latter requiring the insertion of DNA. The ability to edit genes was a major breakthrough in understanding how the system works.

Here is a video from Youreka Science/iBiology that does a really good job at visualizing what happens.

Early Applications

This system is very effective, yielding 90-99% knockdown in gene inactivation studies. Moreover, thus far there has been very few off target effects, meaning that there are no other serious genetic defects other than the ones that scientists are directly causing.

So far, the system has been used to find out downstream effects of genes, again, by detecting what happens in a cell when genes and subsequent proteins are missing or cellular occurrences when agene is overly activated (by 1000 fold).  It has also been used to direct stem cell maturation specifically towards becoming neurons. Larger goals of the utilization of the CRISPR/Cas system though is for developing genetic screening or therapy against viruses and cancer.

Developments

Even though it’s been 20 years in the making, first discovered in the late 1990s/early 2000s and not being used for gene editing until 2013, there is a lot of work still being done to modify the system for better drug therapies. Some current studies are ones using light for more control over when genes are turned on or off and the first human trial to, hopefully, establish a way of ending cancer.

Light Activation

Very recently, scientists at MIT are developing the CRISPR system so to have even more specificity and control by using light. By altering the Cas enzyme, scientists made gene alteration light-inducible and regulatable. To make it light-sensitive, Jain’s lab created what they call “protectors” made of light-cleavable bonds along the DNA backbone that stick to the guide RNA. That way when the right wavelength of light, 365 nm in the UV spectrum, shines on the cell, the DNA breaks off the guide RNA thus allowing for it to bind to the Cas protein.

The way they tested it was with the use of a protein associated with fluorescence, GFP (green fluorescent protein). Basically, when the UV light that breaks the DNA was shown, there was no green glow from the cells. Sangeeta Bhatia, a member of MIT’s Koch Institute for Integrative Cancer Research, explains that “the only targets that were cleaved after light exposure were those being photo-protected” verifying that the light-sensitive DNA protector works.

A very pertinent way this UV light system can be employed is with skin cancer. Because the skin can be easily exposed to ultraviolet light, it may be used to turn off cancerous genes involved in melanoma. In general, the precise control over the exact timing of gene editing is very interesting in itself, and could also shed some light on the timing of cellular events involved in disease or even aging.

Human Trials

Back in July, a team led by Lu You of Sichuan University in China, announced that they would pioneer the first CRISPR trial in humans. Their plan was to remove human T-cells, involved with our immune system, from the bloodstream of patients and knock-out a gene that codes for a protein called Programmed Cell Death Protein 1, or PD-1. This protein is associated with tumors that avoid being processed by the immune system making it difficult for the body to halt cancerous growth. However, the team hypothesized that re-introducing the modified cells using the CRISPR system would act as a solid novel therapy for cancer.

The study started in August and further reports have yet to be made, but overall it has rekindled theethical debate surrounding CRISPR. Because it has the powerful ability to alter genes in any cell, the concern is that it will be used during embryo development or, more extremely, for bioterrorism ends.

In general, the history of genome editing hasn’t been the most glamorous despite how far we have come in scientific and technological advances. But not only is it complicated and fraught with unexpected outcomes, many think that changing DNA, especially in humans, crosses an ethical line and tests on humans should be halted immediately. That being said, the logistics of using CRISPR to make ‘designer babies’ and ‘weapons of mass destruction’ are extremely difficult and fears of such things occurring, at least thus far, are not supported. The real goals of using this system is to get rid of genetic diseases and cure thus far untreatable medical issues. This would allow individuals with multiple sclerosis (MS), for example, not just become healthy and live longer, but to have children that would not have the disease. Moreover, ethical outlines for genetic testing on embryos are in the making.

Final Thoughts

It is very important for scientists to be aware of the ethics of research not only so results aren’t skewed or flawed but also to make sure the tests are humane and morally correct. But the fear of how something might be used should not halt the progress of developing CRISPR at all. All studies have been ethical and non problematic and the results have been overwhelmingly positive with implications of literally curing cancers and genetic diseases. It’s very exciting to see where this research will take humanity, hopefully to healthy longer lives!