CRISPR: what’s the big deal?

By Rich Feldenberg:
In the last couple years there have been a growing number of mainstream media stories (like this recent CNN article) highlighting a new molecular biology technique that is revolutionizing the way scientists conduct genetic experiments, and may soon make the holy grail of medicine (gene therapy) possible. It certainly seems far from usual for the media to be overly concerned with a technical method of scientific investigation, but CRISPR has caught the attention of scientist and non-scientist alike due to its huge potential to change the research landscape. This article will discuss what CRISPR is all about, what it does in nature, how scientists are using it in the lab in place of older more traditional techniques, and what its future potential might be to cure diseases that are now incurable.

CRISPR (pronounced like Crisper) is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats. Yeah, CRISPR is much more fun to say. It is a naturally occurring segment of DNA found in many prokaryotes. Prokaryotes are single celled organisms that lack a nucleus and other internal structures that more advanced eukaryotic cells contain. The prokaryotes include bacteria and archaea, and CRISPR has been found to occur in about 40% of bacteria and 90% of archaea sequenced so far.

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The CRISPR DNA is organized in a particular way. It is made of short lengths of repeating DNA basepairs that are separated by regions of seemingly random DNA, known as spacers. Both the repeating regions and the spacer regions are on the order of 24-48 base pairs in length. These repeating structures were first discovered in the DNA of E.coli (a common bacteria found in the intestines of humans) back in 1987, but their function was not known at that time. It was not until 2005 that their function began to become understood, thanks to bioinformatics. Bioinformatics is a computational biological approach to problems in molecular genetics. Using computer programs to search and compare genetic databases, it was found that the spacer portions of CRISPR exactly matched portions of DNA from bacteria infecting viruses and plasmids. That lead to the realization that CRISPR serves as a kind of immune system for prokaryotic cells.

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Basically what happens is that when a virus infects a bacterial cell, the cells first line of defense includes nucleases (enzymes that cut up DNA) that are released in an attempt to destroy the invaders genetic code. The majority of cells infected will not survive, but in the rare chance that it does survives the viral attack, nucleases then cut up the virus DNA into small parts, some of which become the spacer segments in the CRISPER complex of the bacterial DNA. Next time that the same type of virus infects the bacteria, the bacteria can quickly identify it based on the DNA match between its CRISPR spacer segment and the viral DNA. Enzymes called Cas (for CRISPR associated) are nucleases that will associate with the spacer sequence. They cut the viral DNA at the specific place where the match occurs. This increases the chance for the bacteria to survive the viral attack and confers a kind of immunity to the cell.

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It has been shown that bacteria that contain CRISPR are much more resistant to virus that have DNA sequences contained in their CRISPR spacers, and if those spacers are removed from their CRISPR segments, they lose that resistance. There are certain Cas enzymes that can continue to add new spacers each time they are attacked with new kinds of viruses, and if these types of Cas enzymes are defective or absent, the bacteria can still defend against attack with familiar viruses but are unable to acquire immunity to new ones.

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This certainly seems like good new for bacteria, which are continuously under attack by viruses day in and day out. In fact, it has been estimated that viruses kill 40% of the bacteria in the oceans each and every day. It didn’t take long for scientists to realize that CRISPR could provide an amazing and precise genetic tool. Since the spacer sequence can recognize very specific regions of target DNA and Cas9 (the particular Cas enzyme that is attached to the spacer) can then carry out a seek and destroy mission of the target DNA. It is like the delete key in a word processor program.
It was found that the target DNA didn’t have to be just viral DNA for the process to work. If DNA from a bacteria, animal, plant, fungus, or apparently any organism was inserted into the spacer sequence of CRISPR-Cas9 complex, then a genetic modification could be easily made to that organism. Using CRISPR, genes can be easily and cheaply edited. This system has set in motion a new revolution in molecular biology that has not been seen since PCR (polymerase chain reaction), a technique to amplify DNA was first introduced in the 1980s.

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So what can CRISPR really do in the lab? It can be used to delete specific genes or segments of genes. All you need is a copy of the basepairs that correspond to where you want your deletion in the target organism, and with that and your Cas9 it will find the correct place on the DNA and then cut the DNA at the precise location. In that way you can remove a gene. You can also insert a gene as well. This is done in a similar manner. You have your target DNA picked out and inserted into your CRISPR system, but in addition you need the gene, or segment of DNA you want to insert placed into the cell, as well. The cell’s own repair mechanism will detect the damaged DNA and attempt to repair the break with the added gene. In this way it acts like the cut and paste function of your word processor.
By removing the DNA from the target you are making an irreversible change to the genome of that organism, but it is also possible to use CRISPR to make reversible changes too. This is done by using a defective Cas9 enzyme. The spacer DNA sequence will still seek out and find the desired region of genome, but due to defective Cas9 the DNA will not be cut out. The spacer sequence will still attach to the DNA and block transcription, so is effectively turning off that gene.

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These sorts of techniques can be used to make “knock out” organisms – organisms that lack a particular gene. Knock out mice, for example, are an indispensable tool for understanding how certain genes function in a whole creature, and how mutations in those genes lead to certain diseases. Genes can also be turned on by CRISPR by combining the CRISPR complex with a promotor – a regulatory element that tells the cell to begin transcribing a particular gene.

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This is now allowing labs around the world to investigate disease causing mutations, including cancer biology, at a much faster rate. The techniques are considered relatively easy to use and master, and much less expensive than traditional molecular techniques to achieve similar results.
CRISPR has already been discussed as a potential therapeutic medical intervention, although, the ethics of genetic engineering are still being hotly debated. There was a attempt to use CRISPR to cure the disease hemophilia by a group of Chinese researchers, although they were not successful it is likely only a matter of time before progress along these lines would make similar trials more effective. Right now it is considered by most to be too new of a therapy for clinical medicine, and even if it could be done, some consider tampering with the human genetic code too dangerous. This might especially be true for genetic alterations that would be passed on to subsequent generations beyond the individual being treated. Some worry that it will go beyond just curing disease, and be used by the wealthy to create “designer babies”. Perhaps couples that want taller, stronger, or smarter children would be able to engineer their children to be so. Would that create an even wider divide between the haves and the have nots? These are certainly questions that should be addressed and discussed, but I do hope that fear won’t prevent this technology from reaching it full potential to treat genetic disease.

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Would it really be so bad to have future generations of humans that are more disease resistant (perhaps less prone not only to heart disease, diabetes, and Alzheimers, but less prone towards depression, addiction, or apathy)? What about a future of humans that are more intelligent, more rational, less violent, more compassionate and empathic? Only by being properly informed can we make the best decisions as a society about how to best use such technology.

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References and other reading:
1. CRISPR wikipedia article: https://en.m.wikipedia.org/wiki/CRISPR
2. CRISPR interference wikipedia article: https://en.m.wikipedia.org/wiki/CRISPR_interference
3. “CRISPR/Cas9-mediated gene editing in human triproneulcear zygotes”; Protein & Cell; May 2015, Vol6, Issue 5, pp 363-372. http://link.springer.com/article/10.1007/s13238-015-0153-5
4. “Molecular Biology of the Gene”; 7th Edition, James D. Watson, Cold Spring Harbor Laboratory Press; 2014. Pages 706-712.
5. http://www.geneticliteracyproject.org/2015/06/25/ethical-and-regulatory-reflections-on-crispr-gene-editing-revolution/
6. http://www.nature.com/news/ethics-of-embryo-editing-divides-scientists-1.17131
7. http://www.cnn.com/2015/10/30/health/pioneers-crispr-dna-genome-editing/
8. Neurologica blog article on CRISPR: http://theness.com/neurologicablog/index.php/gene-editing-humans/
9. “Is Bad Luck Really a Diagnosis?”, Darwin’s Kidneys blog: https://darwinskidneys-science.com/2015/06/14/112/

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