How is CRISPR coupled to keeping cells healthy?

The Nobel prizes were announced around a month back, and this year’s prize in Chemistry was special. For the first time since the award’s inception, it was awarded to two female scientists – Jennifer Doudna and Emmanuelle Charpentier, for their invention of the CRISPR gene editing technique which revolutionized molecular biology field. This technique allows one to make specific and precise changes to any part of the genome which can have highly beneficial applications in several areas like - (i) treating diseases, (ii) medicine/drug development, (iii) agriculture. Before this technique was developed, it was extremely difficult to do any kind of genetic modification efficiently and effectively. Thus, suddenly there was an extremely easy option to do any kind of modifications to the genome. It usually takes decades to realize the importance of such techniques but the inception of the CRISPR technology and its applications took a mere 8 years to be awarded the Nobel prize. This is an extremely smart and easy to implement technique but it is not a standalone technique. It relies on the intrinsic DNA damage response pathways of the cell to do its precise editing. Therefore, in this article, I will describe how CRISPR utilizes the cellular DNA repair pathways to perform extremely precise genetic modifications, to fully understand this revolutionary technique.

Each cell of any living organism is made of several DNA molecules, containing all the genetic information of that organism. These are double-stranded helical structures and are made up of different arrangements of four base codes comprising the DNA sequences unique to every organism, and the differences in these sequences lead to the different physical characteristics. These are huge sequences and can vary from millions to billions of bases depending on the organism. To have an idea of the genome size – the bacterial genome usually has a few million bases whereas, in contrast, the human genome has around 3 billion bases. As a small disclaimer: here, I will refer to DNA as the genome interchangeably, which is the collection of all the DNA molecules in a living cell. Proper functioning of all cellular processes requires the genome to maintain its integrity as any changes to it can lead to malfunctioning of various proteins and can eventually lead to various cancers. And to make things worse, the genome is usually under high physical stress all the time - from various DNA binding proteins, external stress, etc. which can easily lead to DNA damages and thus to the loss of genomic integrity. To account for such damaging events, cells deploy several DNA repair pathways that can repair the damaged DNA and return the cell to its normal functioning state. The most important of these pathways is Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). These are highly efficient pathways that keep us free of diseases like cancer. These pathways are usually specific to the different growth stages of a cell but can occur throughout the cellular cycle. Let’s further understand these important processes with more details.

High external stress can easily lead to the breakage of the DNA leading to the formation of free double stranded DNA ends. Both NHEJ and HR can repair these broken ends but with different levels of accuracy. Both these pathways employ different sets of proteins and repair the DNA using very different mechanisms. Whenever the NHEJ proteins encounter such broken DNA ends, they join the two ends without any reference DNA. This is the faster pathway but can also lead to genetic deletions which can be harmful. But as there is not always a reference DNA sequence available throughout the cell cycle, this pathway can be employed whenever such breaks occur. In contrast, HR utilizes a reference DNA template to do the repair. The DNA is replicated in later stages of cell growth and thus only in these stages can a reference template be available for the HR pathway. However, even if HR is not active always, it is highly accurate as it can repair the genome exactly how it’s supposed to be by using the reference template. There are numerous proteins usually involved in either of these pathways and the proper functioning of each of them is extremely crucial for maintaining genome integrity. The schematic below represents how both these pathways repair broken DNA.

Now, as we have a decent understanding of the DNA damage response, let’s head back to the CRISPR editing technique. CRISPR is a form of the immune mechanism used by bacteria to fight bacteriophages or simply phages (viruses which infect bacteria). It was first discovered in the late 1980s but it was not until the 2000s when the functioning of the CRISPR system was understood. Bacteria after surviving a phage infection can store a part of the phage genome in its own genome in between repeated sequences called protospacers. These acquired sequences are unique to the phages and so were not present in the bacteria before the infection occurred. After incorporating these sequences, they actively search for these sequences throughout the cell using several associated proteins called CAS, to detect subsequent phage infection. To simply think about this, such a response is very similar to how the police can look for a known criminal for a similar crime they previously committed. Once such sequences are detected, the CAS proteins can perform cuts on these sequences to counter the phage infection. Thus, this is how bacteria develop immunity against further phage infections.

The CRISPR system was known long before Doudna and Charpentier developed their gene editing technique which earned them the Nobel prize. So, what was so unique in their approach which was not known? Well, even though after extensive knowledge about the CRISPR immune response, how exactly do the CAS proteins find their targets were not properly understood. After discovering that the target search involved small RNAs, in 2012 Doudna and Charpentier showed that by programming any desired sequence in a small RNA molecule called guide RNA (which together with CAS9 can find and bind to its complementary DNA molecule in the genome), precise cuts can be achieved anywhere in the genome. As CAS9 cuts the DNA, several DNA repair pathways get triggered. By introducing a DNA template of choice, the HR pathway can incorporate it into the genome. Thus, precise genetic insertions can now be performed. Also, by introducing multiple cuts, it is possible to perform genetic deletions via NHEJ. Therefore, with this revolutionary work, CRISPR evolved from a mere bacterial immune response to a highly powerful genome editing technique by utilizing the DNA damage response pathways. This work laid the foundation of the whole genome editing revolution as suddenly incorporating changes to the DNA became extremely straightforward. In 2013, Feng Zhang showed that CRISPR can be applied to mammalian cells as well and thereby increasing the technique’s applications further.

CRISPR editing technique finds numerous applications in genetic treatments. But it is still not a perfect technique as off target cuts have been observed to occur in many cases. Hence, extreme precautions should be taken before implementing this technique in live patients as there might be harmful undesired effects. In 2018, such a scenario emerged when He Jiankui used CRISPR to create genetically modified babies. This incident faced widespread criticism raising both health and ethical concerns. But, even with such negative instances, CRISPR continues to push the boundaries of genetic editing and molecular biology. Hopefully, with the ongoing efforts of many research groups, this technique will be perfected soon enough so that it achieves even broader biological and biomedical applications, improving human lives.

Finally, I would like to highlight the importance of basic science research which has made this technique so powerful. Even though CRISPR finds such broad medical and pharmaceutical applications, it was initially studied not as a gene manipulation technique but as an intrinsic bacterial immune response process. Alongside this, CRISPR could be implemented because of our extensive understanding of the DNA repair pathways from several years of studies by numerous scientists. These pathways are so complicated that it is still not fully understood. Without the inquisitiveness of these basic science researchers, we wouldn’t have this technique in existence. Thus, this is the end product of several years of curiosity driven research on several broad and distantly related topics.

I am extremely grateful to The Cosmic Treehouse for giving me this opportunity to write this article. I really enjoyed the entire process. I also want to thank Charu Mehta, Vu Le-Huy Tran, and Shachi Mittal for providing valuable feedback to improve this article.

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