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Genome Editing 101: Gene Editing Techniques

Foreword


For the first time in human history, we not only understand the language of life, but we can also manipulate it. In the past two decades, a lot of advancements have been made to contribute to the realm of genome editing. Genome editing is the combination of techniques that allow us to manipulate the DNA of bacteria, fungi, and even humans. Manipulating genetic codes have an enormous influence and can be used to increase crop yields, produce medicines in bacteria, and even cure genetic diseases.


The Genome Editing 101 series offers to explain the relevant biology of gene editing, the mechanisms by which such techniques work, and the ethical and political problems that arise from this new field of biology. It will be mainly divided into the following chapters:


1. Genome Editing 101: Central Dogma of the Cell


2. Genome Editing 101: Gene Editing Techniques


3. Genome Editing 101: Applications of Genome Editing


4. Genome Editing 101: Politics on Genome Editing


5. Genome Editing 101: Ethical Problems in Genome Editing


6. Genome Editing 101: Future of Genome Editing


Gene editing techniques allow for changes to be made in the genome of a cell. Each gene, or the region that codes for a specific trait, consists of adenosine (A), cytosine (C), guanine (G), and thymine (T). The order of these nucleotides is the code of that gene and is translated into a specific protein. The size of genes is immensely diverse, ranging from less than 100 to over 100 000 nucleotides (Lopes et al., 2021). Changes in genes, or mutations, occur naturally. This is how evolution has shaped the beautiful biological diversity that is present today. If the mutation makes an individual better adapted to its environment, the individual will procreate more and pass on its genes, which gives rise to natural selection (Darwin & Kebler, 1859). Contrastingly, mutations can have adverse consequences, for example causing diseases like cancer that could result in an individual's death.


As sequencing genomes has become cheaper, faster, and more widespread, more diseases are found to have a genetic cause. The first genetic disease identified to be caused by specific mutations was Huntington’s Disease (Gusella et al., 1983). In the HTT gene, there is a tiny sequence CAG, which normally repeats itself between 16 to 20 times. In patients suffering from Huntington’s Disease, this CAG sequence has been found to repeat itself over 35 times. Consequently, the protein that this gene codes for is unstable and aggregates, thereby becoming toxic (Labbadia & Morimoto, 2013). Because the levels of the HTT protein are the highest in the brain, this is where the most damage is done (Schulte & Littleton, 2011).


Figure 1: An overview of the genetic cause of Huntington’s Disease. Because the repetitive sequence CAG is repeated many more times, the protein misfolds and aggregates in the brain cells of patients (Sharman, 2021).

Some genetic diseases have much more subtle causes. Sickle cell disease is one of the most severe diseases caused by mutations in a single gene, and therefore is an example of a monogenic disease (Rees et al., 2018). A single mutation from T to A in the β-globin gene is responsible for a misfolded protein. This causes an erroneous shape of red blood cells, resulting in blood clots that can block smaller arteries (Rees et al., 2018). Because this disease is both severe and caused by a point mutation in only one nucleotide, it is often an ideal target for gene editing tools (Harrison & Hart, 2018).


The first technique that allowed for the manipulation of a cell’s genome was identified in the 70s. In bacteria, specific proteins, termed restriction enzymes, were found to be able to cut DNA at highly specific sequences (Roberts, 2005). As part of the bacterial “immune system”, these enzymes cleave the DNA of viruses, or bacteriophages, that specifically target bacteria. Over the years, more restriction enzymes were identified, and nowadays a complete arsenal is available to cleave DNA at the required sequence. These enzymes are still used a lot in biology, for the synthesis of artificially formed DNA or recombinant DNA (Roberts, 2005).


Restriction enzymes are used to create cloning vectors. These are pieces of DNA that are manipulated and grown in bacteria (Roberts, 2005). By using a restriction enzyme, scientists create so-called sticky ends. As seen in the second step of Figure 2, these sticky ends are small sequences that are not double-stranded. Because the location where each restriction enzyme cleaves DNA is known, a sequence can be inserted into the vector given that the bases on its two ends are complementary to those sticky ends. If that is the case, the foreign DNA segment can be integrated into the vector (Roberts, 2005).

Figure 2: A schematic overview of using restriction enzymes in cloning vectors. The vector is cleaved open by a restriction enzyme (left). The DNA of interest needs to be complementary on both ends to the sticky ends of the cloning vector (middle). The sticky ends in the vector recognise and bind the complementary sequences on the foreign DNA, which is integrated into the vector (BioSite, 2022).

The next major development in gene editing was the identification of Zinc-finger nucleases (ZFNs). In contrast to restriction enzymes, these ZFNs could be synthesised in a lab, and therefore be made specifically for any sequence of choice. Restriction enzymes, on the other hand, are purified from bacteria, and so can only be used to target sequences they evolved to recognise. The development of ZFNs started the real genetic engineering revolution, as it was now theoretically possible to change any sequence (Harrison & Hart, 2018). First, a double-stranded break (DSB) is introduced by these ZFNs. This lesion is repaired by a cellular repair mechanism, termed homologous recombination (HR). The cell repairs this DSB by searching for templates (Cathomen & Keith Joung, 2008). Thus, in patients with sickle cell disease, a T can be given to a cell as a template, then a DSB can be introduced on both ends of the mutated A. This way, the cell will incorporate the T where the A used to be. Applying the same techniques to other diseases with known mutations, gene editing can be a solution to many genetic disorders.


Similar to restriction enzymes, specific ZFN-complexes target specific sequences. For every new sequence to be targeted, new ZFNs need to be synthesised, which is a difficult process (Belhaj et al., 2013). Luckily, a breakthrough was made in 2012 by Jennifer Doudna and Emanuelle Charpentier (Jinek et al., 2012). A complex system, termed clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated domain (Cas), or CRISPR/Cas, had been found earlier that year (Wiedenheft et al., 2012). CRISPR is a system used in certain bacteria to recognise and inactivate viral DNA and RNA based on their specific sequences. There are many different types of this system, and there are many different proteins involved in each of these types. The most interesting type is CRISPR/Cas9. Cas9 is one single protein that binds guide RNA (gRNA). It then searches for complementary DNA. When found, it cuts the DNA at a specific location based on the gRNA, resulting in a DSB. After this, the cell can repair its break by specific mechanisms such as HR (Jinek et al., 2012).


Figure 3: An artwork on Zinc Finger Nucleases. DNA is depicted in red, and the proteins in blue (Goodsell, 2007).

The CRISPR system has evolved to be able to detect and destroy viral sequences before they can do any damage to bacteria. The part of the bacterial genome of importance consists of the CRISPR array and the cas-genes. The former is made up of unique sequences, termed spacers. Between each spacer is a repeat which contains the same sequence, as visible in Figure 3 (Jinek et al., 2012). The cas-genes encode the Cas proteins, depending on which type of CRISPR-system is used. This differs per bacterial species. When an unknown sequence of DNA is found in the cell, this sequence is integrated into the CRISPR array between two spacers. The spacers are then transcribed into sequences that are called CRISPR RNA (or crRNA). There are three types of CRISPR/Cas systems, each of which involves different Cas proteins that collaborate with the crRNA in a different way. The Type II system is the easiest to comprehend and use. In this system, Cas9 binds the crRNA to form a complex which roams free within the cell and induces a DSB when the crRNA finds a matching sequence.

Figure 4: The CRISPR array consists of repeats (R) and spacers (S) that are transcribed, and become the gRNA with which Cas9 combines to demonstrate specificity (Helmholtz, n.d.).

Cas9 is able to distinguish viral DNA from its own DNA by PAM sequences. These are sequences of only a few nucleotides that are very close to the actual target of the Cas protein. In short, two requirements need to be fulfilled for a DSB to be introduced. First, a PAM sequence must be present; and second, the crRNA needs to match (Jinek et al., 2012). The system can be manipulated relatively easily. Scientists can very precisely synthesise gRNA, which is nothing more than an artificial variant of crRNA. Additionally, more PAM sequences have been identified, which can be used with different proteins (Jinek et al., 2012). Through synthesis of specific gRNA, CRISPR/Cas9 can be used to insert or remove a sequence, as demonstrated in Figure 4.


Figure 5: Method of using CRISPR/Cas9. First, the gRNA is synthesised and added to Cas9. When the PAM sequence has been found and the gRNA finds its match, a DSB is introduced, which can lead to a number of consequences. These include the insertion or deletion of a specific sequence (Adjusted from Addgene, n.d.).

Immediately after CRISPR/Cas9 was identified as a possible tool for gene editing, the power of this technique was recognised. The major advantage of this system is that a single protein, Cas9, does the work that many subunits of ZFNs do (Belhaij, et al., 2013). Cas9 is therefore much simpler in terms of usage. Additionally, by giving Cas9 a specific sequence of gRNA, researchers can very easily target a sequence and incorporate a new one meant for insertion. The cell can then deploy HR mechanisms to allow for a knock-in, i.e., the insertion of a specified sequence (Belhaj et al., 2013).


Over the last decade, CRISPR/Cas9 has caused a revolution in gene editing. Consequently, the Nobel Prize for Chemistry was awarded to Doudna and Charpentier, the founders of CRISPR technology, in 2020. Interestingly, Doudna saw this revolution coming, which is why, in her 2015 TED talk, she tried to bring out a message to the general public regarding the ethical questions that gene editing, and CRISPR/Cas specifically, evoke (Doudna, 2015). Similarly, another scientist who pioneered the CRISPR technique, Michael Willes, is very clear in an interview he gave in 2016 (Cohen, 2016). He emphasised that the revolutionising aspect of this technique is the ease with which it is deployed, to a point where "any idiot can do it” (Cohen, 2016). Furthermore, CRISPR is not only easier to use or more reliable, but it is also significantly cheaper than its alternatives (Cohen, 2016). Though certain skills and knowledge are still required for the synthesis of gRNA, among others, any scientist somewhat familiar with the field now has the means to edit a genome.

Figure 6: The number of publications per year using the PubMed search term ‘CRISPR Cas9’, showing the rapid development of CRISPR technology in the past decade (Harrison & Hart, 2018).

For the first time, humankind is able to manipulate practically any genetic information with incredible precision and efficiency (Doudna, 2015). The discovery of CRISPR/Cas9 further allows research to edit genomes in a cheap and easy manner. By implementing a specific RNA sequence, Cas9 can be led to target virtually any piece of DNA. In contrast to earlier gene editing techniques, CRISPR/Cas9 only requires the manipulation of a single protein, which is precisely what makes this technique so easy to use. Because the CRISPR era is still young, the field is still developing. Various technical issues, e.g., off-target effects (Listgarten et al., 2018), still require robust research and improvement, not to mention the emergence of increasing ethical concerns. The applications of gene editing, as well as its development in political and ethical fields, will be discussed in future articles of this 101 Series.

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