The Background and Applications of CRISPR/Cas9 Technology

By Reagan Smith


CRISPR/Cas9 is the latest gene editing technology in the world of science. It was first discovered in the Escherichia coli (E. coli) genome by Francisco Mojica in 1987; however, the modern day application of this mechanism was not realized until 2008. In nature, bacteria utilize the CRISPR/Cas9 complex to protect themselves against invading viruses. This process begins when the bacteria detects any sort of viral DNA. Once they do this, they produce two types of short RNA (a single stranded form of genetic information, similar to DNA), one that matches the viral DNA sequence and one that compliments it. These 2 short RNAs bind to a Cas9 protein (one of many CRISPR associated proteins), which has the ability to cut the two strands of DNA through a double stranded break (DBS). The Cas9 protein then cuts the viral DNA where the matching RNA binds, rendering it ineffective and disabling the virus. In its entirety, CRISPR is made of a protein molecule and a guiding RNA strand (gRNA) that binds to the target DNA section of the genome.



Image Credit: Flickr @ Francisco Gonzales

In 2008, scientists discovered how to use CRISPR to not only cut DNA, but to also replace the cut DNA with a specific sequence via homology directed repair. In this process, cells repair the break in the DNA by utilizing sequences found nearby. Scientists can also engineer Cas9 to cut at any location in the genome by changing the gRNA in the CRISPR complex. Essentially, CRISPR can be used to edit the genome of any species on Earth, including humans. Because of its versatility, simplicity, and low cost, CRISPR is arguably the largest technological jump in the world of biology thus far.


Image Credit: Flickr @ NIH Image Gallery

In addition to this, CRISPR can be used to modify epigenetics—a way of influencing gene expression without changing the actual nucleotide sequence. Scientists are able to do this by using CRISPR/Cas9 to study the targets of epigenetic markers, including DNA methylation or acetylation at specific locations in the genome. For example, researchers used CRISPR to knock out all three active DNA methyltransferases—enzymes that transfer a methyl group to DNA to repress its transcription—to study their impact on gene expression in embryos. In the future, scientists may be able to utilize CRISPR to specifically activate or deactivate epigenetic markers and alter gene expression, which they can then link to cellular processes such as cellular differentiation or development of diseases.


Currently, many pharmaceutical and biotech companies are creating a slew of CRISPR-based genetic therapy treatments to correct mutations in patients’ cell genomes, a treatment plan that, if successful, would eliminate the possibility of a patient’s body rejecting donor organic material. In this process, target cells are removed from the patient’s body in order to test out CRISPR on the genome as well as to ensure that no additional mutations occur. Then, the technology is applied to the entire target tissue or organ to fix the mutation causing the disease.


What makes CRISPR/Cas9 even more interesting is the fact that it can not only trigger cell differentiation, but can also create specific types of tissues, which can then be transplanted into the patient. Utilizing this technology, scientists have transplanted muscle cells to treat muscular dystrophy and hematopoietic stem cells in order to treat sickle cell anemia.

The CRISPR/Cas9 system can also be turned into a “gene drive,” meaning that it transfers disease-resistant genes, such as those that can deactivate malaria, to populations. This system is modified by putting disease-resistant genes with CRISPR target RNA and Cas9 proteins, and this group then inserts itself into parental chromosomes before being passed onto offspring.


Looking beyond treating humans, CRISPR can also be put to use in ecological settings. Scientists are examining how to bring back extinct species by placing their DNA in a closely related organism to “recreate” that extinct animal, which could then interbreed and increase the abundance of that species. Alternatively, we can mutate invasive species’ DNA to render them unable to survive, a process that could drive these species to near extinction in areas and reduce their negative impact on the ecosystem.


With this powerful technology comes risks, however, and one experiment in recent years stands out as a questionable example of the application of CRISPR. He Jianku, a scientist from Shenzhen’s Southern University of Science and Technology, used human germline genome editing to genetically modify embryos before placing them in women’s uteruses to be born. Human germline genome editing varies from other gene therapies in that the modifications induced by CRISPR in embryos affect somatic and sex cells, meaning sperm and egg cells, and these modifications will be inherited by progeny. There are a couple different ways to edit the germline, including editing an early-stage developing embryo, where changes to cells manifest in gametes, editing gametes before fertilization occurs in vitro, and editing the genome of eggs or sperm-forming cells in babies, children, and adults.

Jianku edited the CCR5 gene that codes for the C-C chemokine receptor type 5, with the goal of protecting the future babies from HIV. He injected the embryos with a CRISPR complex designed to delete 32-bp in the CCR5 gene to produce non-functional CCR5 proteins. However, normal CCR5 proteins are found on all T-cell surfaces (white blood cells in the immune system) and may be involved in brain mechanisms, meaning their deletion could be destructive. This experiment resulted in one pregnancy, producing two non-identical twins, named Nana and Lulu. Jianku’s addition of CRISPR did not produce the predicted modification, however; one twin had only one copy of their CCR5 allele modified, while the other copy of the allele remained normal. The other twin’s CCR5 alleles mutated in an unplanned way, and Jianku lost control of the intended mutations. Overall, CRISPR did not make the intended modification of the 32 bp deletion, instead it made other changes that produced the end result of a non-functional protein, but these changes were entirely new in humans.


Investigation into the safety and validity of this experiment has been largely unsuccessful. Jianku’s university had previously posted informed consent documents, but those, as well as any pages related to him and his research, have been deleted and removed from the website.


Looking back, this experiment demonstrated a need for regulation of CRISPR/Cas9 to the scientific world, which has already formed various committees and groups to discuss and create guidelines. We are examining an interesting intersection between science and ethics, and while the field is limitless, we need to ensure our progress is positive, not negative. Overall, CRISPR has endless applications that can increase our lifespan, stop debilitating diseases, and heal ecosystems, and one can only imagine the impact it will have on the human race’s future.


Educational Content

  1. What forms the CRISPR/Cas9 complex?

  2. It consists of a protein bonded to a guide RNA whose base pairs compliment the target DNA strand, as well as a nuclease protein that cuts the double stranded DNA at a specified location

  3. What are a couple applications of CRISPR?

  4. Scientists can affect epigenetic markers, like acetyl or methyl groups, on specific locations in DNA to “map” those markers and study their effect. They can knock out mutated DNA sequences causing harm to patients and replace those sequences with a different strand to treat diseases. We could, with careful regulation, even preemptively treat genetic diseases in embryos, as He Jianku attempted to do. Finally, scientists could potentially insert an extinct animal’s DNA into a related organism via CRISPR, thus bringing that species back from extinction. Vice versa, they can negatively modify a harmful invasive species’ genome to render it nearly extinct and reduce its impact on the local ecosystem.


Sources

https://www.genscript.com/applications-of-crispr.html

https://www.youtube.com/watch?v=2pp17E4E-O8

https://www.youtube.com/watch?v=pVIVSpUgR44&feature=emb_title

https://academic.oup.com/jlb/article/6/1/111/5549624?login=true


Image Credit:

No changes were made, TAGTAT | Cosmocaixa Barcelona. Spain used by: theconversatio… | Flickr, License: Creative Commons Legal Code

No changes were made, CRISPR Cas9 | CRISPR-Cas9 is a customizable tool that lets s… | Flickr, License: Public domain

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