Cutting Edge: CRISPR/Cas9’s Molecular Scissors

Hazel Walker explores the Nobel Prize-winning innovation of CRISPR/Cas9 gene editing

On the 7th October 2020, the news broke that the Nobel Prize in Chemistry had been awarded to Professors Emmanuelle Charpentier and Jennifer Doudna for the ‘development of a method for genome editing’. Only eight years after their discovery that the CRISPR/Cas9 system can be used to edit genes, it is hailed as one of the most impactful scientific breakthroughs of the 21st century. Notably, this was the first time the prize had been awarded solely to two women with Charpentier telling the Nobel Prize Committee, ‘My wish is that this will provide a positive message to the young girls who would like to follow the path of science, and to show them that women in science can also have an impact through the research that they are performing’.

Like many scientific breakthroughs, the story of this genome-editing discovery started with an unexpected observation. In 1993, Francisco Mojica, a doctoral student based in the University of Alicante, noticed a curious repetitive sequence of DNA in the genome of the bacteria he was studying. Upon realising that these repetitive sequences of DNA were present across many strains of bacteria, and had first been reported in 1987, Mojica and other scientists set to work on understanding their purpose. They concluded that the stretches of DNA, which they named Clustered Regularly Interspaced Short Palindromic Repeats or ‘CRISPR’, were part of a defence system to protect micro-organisms such as bacteria and archaea from viral infection.

Like us, bacteria are susceptible to viruses, called bacteriophages. When a bacteriophage infects a bacterial cell, some of the foreign bacteriophage DNA is integrated amongst the repetitive CRISPR sequences. This forms a sort of ‘immune memory’, similar to how the human immune system can remember a prior infection and more effectively defend against it a second time. The stored bacteriophage DNA is transcribed to a messenger molecule called RNA, which can then recognise and bind to its complementary sequence, should this particular bacteriophage attempt to invade the bacteria again. The CRISPR RNA also brings with it a CRISPR-associated protein, ‘Cas9’. Cas9 acts as a pair of scissors, chopping up the incoming foreign DNA and stopping the infection in its tracks. This step forward in the understanding of bacterial defence was exciting in itself, as it was the first time that immune memory had been demonstrated in bacteria. However, CRISPR/Cas9’s potential as a revolutionary genome editing tool was yet to be realised.

In 2011, Emmanuelle Charpentier, an Associate Professor at Umeå University in Sweden, added another piece to the puzzle. She found that a second type of RNA, ‘transactivating RNA’ or ‘tracrRNA’, was required for CRISPR RNA to take its mature form. Shortly after this, Charpentier started a lifechanging collaboration with University of California, Berkeley biochemist Jennifer Doudna. Charpentier and Doudna hypothesised that, if they tweaked the sequence of CRISPR RNA to match a target of their choice, they would be able to cut DNA at selected sites. They were right, publishing their ground-breaking work in Science in 2012.

The breaks in DNA that the Cas9 scissors introduce are key to the system’s potential as a genome editing tool. When DNA is cut, it must be repaired. Fortunately for those hoping to edit genes, the DNA repair mechanisms employed by cells are error prone. This means that the resulting repaired DNA sequence is often incorrect. If there are too many mistakes in the genomic DNA, the resultant protein will be nonfunctional, essentially causing it to be switched off. Genes can also be edited by introducing extra DNA to the cut site, allowing for precise modification of the DNA sequence within the gene.

The possibility to have such precise control over genetic sequences expanded the existing genome editing toolkit and quickly became a widely used method in research laboratories due to its efficiency and low cost compared to previous methods. Researchers can even buy pre-designed CRISPR RNAs or use simple software to design their own, drastically speeding up the genome editing process.

With a tool that can permanently switch off a chosen gene, researchers can study the effect that its loss has on biological processes in model organisms, unpicking the role of the gene in health and disease. Furthermore, researchers can use CRISPR/Cas9 to tag proteins of interest with geneticallyencoded fluorescent probes which, when viewed under a microscope, can reveal information about the expression pattern and function of that protein.

Perhaps the biggest impact CRISPR/Cas9 could have is the treatment of genetic diseases. Shortly after the landmark Science paper, the CRISPR/Cas9 system was used to successfully edit mammalian and even human cells. The power of this technology for editing the human genome was evident and the moral and ethical implications of this were worrying. In 2015, many prominent researchers called for a global moratorium on the use of the technology to edit human germline cells (eggs, sperm, and viable embryos) in which potentially unknown side effects could be inherited by future generations. While this moratorium from respected scientists proved a deterrent for most, CRISPR hit the headlines in 2018 when Chinese researcher, He Jiankui, announced the birth of the world’s first CRISPR edited babies. Jiankui had convinced a couple undergoing in vitro fertilisation (IVF) that their embryos could be edited to be immune to HIV, sparking widespread controversy, outrage, and condemnation from both the scientific community and the public. Whether the babies are actually immune to HIV and do not suffer from side effects still remains to be seen.

Currently, the safety and efficacy of CRISPR/Cas9 genome editing is being tested in tightly regulated human clinical trials, targeting non-germline cells to treat diseases such as cancer, blood disorders and blindness. In most cases, a patient’s cells are removed from their body, edited, and then re-introduced — which is likely to be safer than injecting the CRISPR components straight into the body. A 2019 trial involving three cancer patients confirmed for the first time that a treatment of this kind was safe and feasible in humans, although it did not improve the patients’ cancer prognoses. In March 2020, researchers moved one step further, injecting the CRISPR components directly into patients for the first time in order to treat hereditary blindness. Administered via the eye, this treatment aims to rectify the mutation that causes a condition called Leber Congenital Amaurosis 10. The trial is expected to conclude in 2024.

CRISPR also has the potential to be an important tool for agriculture. CRISPR-induced changes can accelerate the traditional process of selective breeding. As the world’s population increases, crops edited to withstand extreme climate conditions could prove key for global food security. Researchers are also working to generate more nutritious crops or those that lack common allergens such as the protein responsible for gluten intolerance and coeliac disease. UK company Topic Bioscience has even developed decaffeinated coffee beans, cutting out the lengthy and expensive process required to decaffeinate normal coffee beans.

In their quest to understand bacterial immunity, researcher’s curiosity and creativity brought us a powerful new technology. A mere eight years after the development of CRISPR/Cas9-mediated genome editing, the possibility of drought-resistant crops and cures for disease are closer than ever. Only time will tell what impact this exciting technology will have on the future but, if its first few years are anything to go by, it looks like it will be world-changing

Hazel Walker is a 4th year PhD student in Immunology at Fitzwilliam College. Artwork by Eva Pillai.