Chemisch Journaal

In 2020 the Nobel prize for chemistry was won by Emmanuelle Charpentier of the Max Planck Unit for the Science of Pathogens in Berlin (Germany) and Jennifer A. Doudna of the University of California in Berkeley (USA). They won the prize for their research on CRISPR-Cas9. CRISPR is a method of removing, adding and Firure 1: Alfr. Nobel medallion replacing DNA to manipulate the genomes of cultured cells, animals, and plants. [1,2] To help you understand CRISPR a little better, five questions about CRISPR and CRISPR -Cas9 will be answered in this article.

What is CRISPR-Cas9 and what is CRISPR?

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.[3] CRISPR-Cas9 has been discovered in the immune system of bacteria, it is an enzyme that cuts double stranded DNA at a targeted location. The CRISPRCas9 complex consists of two main parts: the DNA unwinding and cutting protein called Cas9 and an RNA part functioning as a guide.[4,5]

By inactivating the Cas9 cutting domain and adding deaminase enzymes the CRISPR complex can perform precise gene editing. The CRISPR complex can edit just one base in the DNA sequence. This precise gene editing disease causes sequences to be deactivated without messing with the rest of the harmless DNA. By adding different proteins to the CRISPR complex gene transcription and silencing can be accelerated.[4] The CRISPR complex can be edited in many other ways. Resulting in CRISPR-systems that can remove, add and replace part of the DNA.

Firure 2: Emmanuelle Charpentier and Jennifer Doudna What was the research of Charpentier and Doudna?

When doing research on bacteria, Charpentier discovered the tracer-RNA (tracrRNA) in bacteria’s ancient immune system. She found out this was part of the CRISPR-Cas system that disarms viruses by cutting their DNA. This finding was unexpected, but she continued working on CRIPSR-Cas in her further research. After the discovery she started collaborating with Doudna, who had been doing research about RNA for a long time. The two bundled their knowledge resulting in a major breakthrough in the scientific world. They succeeded in recreating the bacteria’s CRISPR-Cas system and even making it more easy to use. This resulted in the sharpest tool in gene editing technology. [6]

The CRISPR-Cas system was edited in such a way that it was able to cut double stranded DNA at a targeted place. While the CRISPR-Cas system in the bacteria was only able to cut the DNA of certain viruses and inactivating them, the CRISPR-Cas9 system was able to cut any DNA.[3] This way it is possible to rewrite the genetic code at any place in the DNA.[6] This discovery made the editing of the human genome possible, which is something scientists have been working on for a long time. The tool has contributed to important discoveries in cancer research and research about curing inherited diseases.[6] Besides, the CRISPR-Cas tool can also be used in agricultural research, creating crops that can withstand diseases and extreme weather conditions.[7]

Firure 3: 3D model of the Crispr-Cas9 system

How does the CRISPR-Cas9 system work?

The CRISPR-Cas9 system contains of a Cas9 protein containing CIRSPR-RNA (crRNA). The Cas9 has helicases to unwind DNA and nucleases to cut DNA. The CRISPR part contains the information on where the cut should be made. The CRISPR part has crRNA that matches up with the corresponding DNA. In the CRISPR-Cas9 system that was found in the bacteria the crRNA is base paired to the tracrRNA, this holds the crRNA in place. In the CRISPR-Cas9 system created by Doudna and Charpentier the crRNA is attached to tracrRNA, forming a hairpin.[2]

In the CRISPR-Cas9 system that was found in the bacteria, the crRNA repeats were fixed sequences that could read the genetic code of viruses. Charpentier and Doudna replaced this RNA with the specific RNA sequence that corresponded to the targeted Firure 4: Bacteria DNA. The CRISPR-Cas9 system under microscope could now be used to cut any DNA at the desired place. The combination of the trcrRNA-crRNA was called guideRNA (gRNA). Charpentier and Doudna called this CRISPR part of the system the gRNA Chimera.[3] A Chimera is a mythological animal that is a combination of a variety of different species. Is it possible to manipulate the DNA of humans by using CRISPR?

Yes, CRISPR can be used to manipulate human DNA. This is possible in humans of any age and in embryos of unborn babies. An example of the first is the therapy of Victoria Grey’s sickle cell disease. This is a genetic blood disorder that causes patients a lot of pain. Victoria was the first person in the world to receive a CRISPR treatment for her genetic disease. [7]

Firure 5: Removing part of the DNA helix

The DNA manipulation of unborn babies is a little bit more of a sensitive topic. Researchers and news media like to refer to these gene-edited babies by using the term “designer babies”. In theory CRISPR-Cas creates the possibility to change the eye color of a baby or the thickness of their hair. But the CRIPR-Cas method can also be used to make the babies immune to certain diseases.

In 2018 the Chinese researcher De He Jiankui came forward with the very unexpected news that he created the first two human designer babies: twins named Lulu and Nana. The genes of the embryos of the babies were edited so the babies would be immune to HIV. Many researchers in the field knew creating gene-edited humans would be possible, but no-one expected this to happen so soon. Mainly because of the bioethical controversy around these designer babies. [4] How could CRISPR help with COVID-19?

Doudna and many other scientists in the world are exploring the possibility to use CRISPR-Cas to make a quicker and more accurate test for COVID-19. CRISPR-Cas13 is used for this purpose, instead of the previously described CRISPRCas9. This system has a fluorescent marker that is cut of the Cas13 protein and becomes fluorescent when COVID-19 is detected. [8]

In conclusion, the CRISPR-Cas9 system is found on accident by Charpentier in her research on bacteria. After rebuilding the complex, she and Doudna created the sharpest gene editing tool available. This finding creates many possibilities in curing diseases, boosting agriculture and many other purposes. The finding is relatively new, so it is to be seen what the future holds for this groundbreaking solution in gene editing.

References

[1] Youtgenome, (2016, December 19), What is CRISPRCas9? Retrieved from yourgenome website: https://www. yourgenome.org/facts/what-is-crispr-cas9 [2] Knott, G. J., & Doudna, J. A. (2018). CRISPR-Cas guides the future of genetic engineering. Science, 361(6405), 866–869. https://doi.org/10.1126/science.aat5011 [3] Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 337(6096), 816–821. https://doi.org/10.1126/science.1225829 [4] Nature Video (2017), CRISPR: Gene editing and beyond, Retrieved from https://www.youtube.com/ watch?v=4YKFw2KZA5o [5] Makarova, K. S., Haft, D. H., Barrangou, R., Brouns, S. J. J., Charpentier, E., Horvath, P., … Koonin, E. V. (2011). Evolution and classification of the CRISPR–Cas systems. Nature Reviews Microbiology, 9(6), 467–477. https://doi.org/10.1038/ nrmicro2577 [6] The Nobel Prize in Chemistry 2020, (2020, October 7), Retrieved from NobelPrize.org website: https://www. nobelprize.org/prizes/chemistry/2020/press-release/ [7] Doudna, J. A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213), 1258096–1258096. https://doi.org/10.1126/science.1258096 [8] Fozouni, P., Son, S., Díaz de León Derby, M., Knott, G. J., Gray, C. N., D’Ambrosio, M. V., Zhao, C., Switz, N. A., Kumar, G. R., Stephens, S. I., Boehm, D., Tsou, C.-L., Shu, J., Bhuiya, A., Armstrong, M., Harris, A. R., Chen, P.-Y., Osterloh, J. M., Meyer-Franke, A., … Ott, M. (2021). Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy. Cell, 184(2), 323-333.e9. https://doi. org/10.1016/j.cell.2020.12.001