An Overview of CRISPR-Cas9 Gene Editing and its Potential in Treating Cystic Fibrosis

Rainbow Zhao ([email protected]) and Aidan Fu ([email protected]) 

1. Introduction

With the current advancements in technology, gene editing will no longer be science-fiction in the near future. Gene editing, in simple terms, refers to the ability to alter a living organism’s DNA and hence, alter certain heritable traits with the help of technology (World Health Organization, 2023). CRISPR-Cas9 is one example of such technology with a great potential in treating patients with cystic fibrosis.

2. Cystic Fibrosis

Cystic fibrosis is an autosomal recessive genetic disorder occurring on chromosome 7q32 (Schwarz and Staab, 2015; Johns Hopkins Cystic Fibrosis Center, 2019). The pulmonary ionocytes located in the bronchiole express the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel at a high level (Luan et al., 2024). These channels are in charge of secreting anions such as chloride ions into the airway (Basics of the CFTR Protein, 2024). In normal situations, chloride is able to exit into the airways through CFTR channels, and water follows through osmosis (ibid). This leads to normal mucus formation. 

On the other hand, patients with cystic fibrosis have CFTR channels with significantly reduced activity (Johns Hopkins Cystic Fibrosis Center, 2019). In this case, chloride is trapped in the pulmonary ionocytes and hence, water cannot exit into the airway through osmosis. Additionally, water enters the ionocytes instead through osmosis. This results in thick and sticky mucus, which immobilizes cilia on ciliated cells (The Cystic Fibrosis Center at Stanford, 2024). Without cilia sweeping and cleaning the airway, the patient is a lot more susceptible to bacterial infections in the lungs (ibid). The thick mucus also obstructs airflow, resulting in patients having trouble breathing (ibid). Other symptoms include nagging cough, frequent wheezing, and slow growth (Cleveland Clinic, 2021).

Figure 1: a diagram comparing the normal cross section of airway with cross section of airway with cystic fibrosis (adapted from: Pathology Project – Cystic Fibrosis 2011)

3. The Natural CRISPR-Cas System

The natural CRISPR-Cas system is actually part of 40% bacteria’s and almost all archaea’s immune defense (Jiang and Doudna, 2015). When a bacteriophage invades and releases its DNA, that DNA can get recognized by the Cas1-Cas2 complex (Lee et al., 2019). Cas1 is an integrase and nuclease, whereas Cas2 is a structural protein (Fagerlund et al., 2017). In short, the Cas1-Cas2 complex is responsible for the scission of parts of the viral DNA (Doudna Lab, 2024). That part of the viral DNA is termed ‘protospacer’, and it’s unique to that specific virus (ibid).

Afterwards, the Cas1-Cas2 complex will take the protospacer to the CRISPR array (Doudna Lab, 2024). The word ‘CRISPR’ refers to a region of a gene found in the bacterium or archaeon’s nucleoid (Smith, 2023). It stands for ‘Clustered regularly interspaced palindromic repeats’, translating to ‘short, repetitive DNA sequences with partially dyad symmetry, scattered among other unique DNA sequences, located at a specific region of the genome’ (ibid). In other words, the CRISPR array consists of repeats and spacers (Karginov and Hannon, 2010). Spacers are those protospacers integrated by the Cas1-Cas2 complex. There is a repeat in between every spacer (ibid). 

Whenever the same virus infects the bacterium or archaeon again, the entire CRISPR array will be transcribed (Natural functions of CRISPR-Cas, 2024). It will later get processed by other enzymes such as Cas6, and eventually will end up with parts of the repeat and the virus’ corresponding spacer (Wakefield et al., 2015). This forms a mature crRNA-tracrRNA complex (Karvelis et al., 2013). The repeat section of the crRNA-tracrRNA duplex binds to Cas9 and activates it, and the spacer pairs up with the viral DNA (Liao and Beisel, 2021). The Cas9 nuclease can then break the viral DNA if it enters the bacterium again.

Figure 2: a diagram illustrating the mechanism of the natural CRISPR-Cas system in bacterial immune defenses (adapted from: Natural functions of CRISPR-Cas 2024)

4. CRISPR-Cas9 in Mammalian Cells

4.1 Delivery

Because the CRISPR-Cas system isn’t naturally utilized by mammalian cells, scientists need to introduce several components of this system into mammalian cells through technology. Among many techniques used, plasmid transfection seems more popular. Firstly, edited plasmids containing the Cas9 gene, sgRNA, promoter, and selection marker are transformed into Escherichia coli (Widney et al., 2024). E. coli is then allowed to reproduce in a nutrient rich environment, so more of the edited plasmids can be produced (Fakruddin et al., 2013). After a few generations, the E. coli are lysed in alkaline solutions in order to extract the plasmids (Delane et al., 2018). Finally, the solution containing plasmids are purified and ready for transfection into mammalian cells (Potter and Heller, 2003). Other techniques include viral transduction and the direct delivery of mRNA and sgRNA (Yip, 2020).

There exist plenty of ways to transfect plasmids into mammalian cells, including lipofection, electroporation, and microinjection (Kim and Eberwine, 2010). Lipofection utilizes lipoplexes, basically a phospholipid membrane surrounding the plasmids (Zhang et al., 2012). The lipoplex can either fuse with the cell membrane and release the plasmids into the cell or taken in entirely through receptor-mediated endocytosis (ibid). Electroporation is accomplished through submerging the cells in a conductive solution (Grys et al., 2017). When electricity is applied, temporary pores in the cell membrane are created (Gehl, 2003). Since nucleic acid is negatively charged, they get attracted to the slightly more positive environment inside the cell. And finally, microinjection involves directly injecting the plasmids into the nucleus of the cell through a pipette (Dean and Gasiorowski, 2011).

4.2 Cas9 and DSB

Now that the genetic material that codes for the Cas9 nuclease and sgRNA are inside the nucleus of the cell, they can be processed into the Cas9 nuclease and sgRNA. sgRNA stands for ‘single guide RNA’ (Costa et al., 2017). It is essentially a synthetic RNA consisting of a spacer sequence and a scaffold region that mimics the natural crRNA (Asmamaw and Zawdie, 2021). After the sgRNA uses the scaffold region to bind to and activate the Cas9 nuclease, Cas9 starts moving along chromatins to look for base pairs complementary to those in the spacer sequence (Feng et al., 2021). When the Cas9 nuclease finds its target, in other words, after the spacer sequence unwinds and pairs up with another DNA strand within the genome, Cas9 performs a double-strand break (DSB) 3 nucleotides upstream of the protospacer adjacent motif (PAM) region (Xue and Greene, 2021). This results in blunt end cuts, allowing the insertion of desired DNA (ibid).

Figure 3: a diagram representing the mechanism behind Cas9 nuclease inducing DSB (adapted from: An Analysis of SARS-CoV-2 on the Molecular and Subatomic Levels through Applied I-Theory 2021)

4.3 ssODN and HDR

Once the DNA is cut, the cell will automatically try to repair it. There are 2 main methods to repair the damage: non-homologous end joining (NHEJ) and homology directed repair (HDR) (Mao et al., 2008). When using the CRISPR-Cas9 technique, HDR is preferred as it allows for precise editing while NHEJ is very error-prone, however, NHEJ is much more efficient compared to HDR (Mao et al., 2008). The main difference is that HDR relies on a template strand. This can be a donor DNA or DNA from the same gene of the homologous chromosome (Alberts et al., 2017). In the case of cystic fibrosis, usually single-stranded oligodeoxynucleotides (ssODN) are used (Smirnikhina et al., 2020). They are synthetically designed to serve as a template for the desired edits (Asmamaw and Zawdie, 2021). Methods of delivery into the cell include electroporation and microinjection (Lino et al., 2018). 

The specific mechanism of using ssODN to edit a gene can vary slightly for cystic fibrosis depending on the context such as the type of cell. One mechanism involves exonuclease activity (Ensinck et al., 2021). In this model, enzymes with exonuclease activity can create short single-stranded overhangs just upstream and downstream of the same strand of DNA (Lovett, 2011). This exposes a few bases on the complementary strand (ibid). ssODN can pair up with these bases and hence, integrate itself within the gene (Levi et al., 2020). Finally, DNA polymerase adds the bases complementary to ssODN on the complementary strand, and ligase seals the fragments (Nambiar et al., 2022). 

The alternative model doesn’t involve exonuclease activity. In this model, ssODN essentially serves as a template strand for the template strand. This ssODN is longer than the DNA segment intended to be inserted at the DSB site, extending a few nucleotides upstream and downstream to pair with one of the DNA strands (Boel et al., 2018). After ssODN pairs up with the DNA strand, DNA polymerase can add the bases complementary to ssODN on the DNA strand (ibid). This complete DNA strand now becomes the template strand for the other. At this point, the ssODN is no longer useful and therefore, is removed (ibid). Meanwhile, DNA polymerase uses the synthesized template strand to complete the remaining strand (Alberts et al., 2002). Ligase seals the fragments once this process is complete (Sizova et al., 2021).

4.4 Fate of Components

The integration of the edits marks the completion of this process. Now, the gene containing the edits can be expressed to influence a trait. However, it is also important to acknowledge the components used during the edit are still inside the cell. 

If plasmid transfection is used, the plasmids will remain in the nucleus of the cell after editing. This means that Cas9 nuclease can be made multiple times even after the edits are made (Lino et al., 2018). However, because the plasmids aren’t a part of the genome, they won’t replicate, therefore, will be diluted after a couple of cell cycles (Dewan and Uecker, 2023). If other methods such as direct delivery of mRNA and sgRNA are used, the RNA will eventually be degraded by exonuclease activity (Pelea et al., 2022). This is also the case for ssODN (Hall et al., 2018).

5. Effect of CRISPR-Cas9 on Cystic Fibrosis

Editing enables the patient to acquire a functional CFTR allele, which allows for the production of functional CFTR channels (Bulcaen et al., 2024). Consequently, this permits chloride to exit ionocytes (Scudieri et al., 2020). The release of chloride ions will naturally be followed by osmosis, and as an effect, the mucus of the patient will become normal (Hanssens et al., 2021).

6. Potential Challenges of Using CRISPR-Cas9

6.1 Applications In Vivo

As of now, most of the experiments done to test the effect of CRISPR-Cas9 in gene editing is done in vitro in a limited number of mammalian cells (Mehravar et al., 2019). Preclinical trials are currently in progress, but it might take more time before clinical trials are conducted on a broader scale. 

To successfully implement this technology in vivo, several complications have to be addressed. For example, the delivery of the components used for this technology can be complicated in vivo. This is due to the involvement of other systems in the body. The components used will most likely have to be incorporated into drugs. There are four main methods of drug administration, including oral, rectal, parenteral, and inhalation (Kim and De Jesus, 2023). All the methods mentioned above involve other systems such as the gastrointestinal tract, circulatory system, and the respiratory system (ibid). Another potential problem is that the use of certain methods for delivering the components such as viral vectors may trigger immune responses, and lead to counter-intuitive situations. Therefore, figuring out a way for the components to successfully reach the target cells exclusively still remains a challenge (Patsal et al., 2019).

Moreover, the other genes in the cell may be susceptible to off-targeting modifications (Blattner et al., 2020). This implies that the sgRNA may still bind to sequences with partial or imperfect matches, and Cas9 nuclease may still induce DSB in these genes (Zhang et al., 2016). Because a donor DNA like ssODN isn’t available nearby, this may lead to the cell using NHEJ to repair instead, which has a high chance of causing random base insertion, deletion, or substitution (Zhang et al., 2021). This will disrupt the reading of codons by ribosomes, and hence, may lead to non-functional or faulty proteins (Allan Drummond and Wilke, 2009). Depending on the gene affected, the cell may lose its function, or may even lead to tumor formation (Cooper, 2020). For this reason, it is extremely important to explore ways to mitigate the risks.

Additionally, the ionocytes in a patient’s body are already differentiated. This suggests that cystic fibrosis can’t be cured by editing the gene of one single somatic cell. To combat this issue, the stem cell of the patient is extracted and edited (Hoang et al., 2022). Then, the stem cells are transplanted back into the patient and allowed to differentiate into ionocytes (King et al., 2020). However, an issue of this is that the ionocytes with cystic fibrosis are still present, therefore, this method is only enough for the symptoms to be improved instead of cured. Ideally, CRISPR-Cas9 should be used on embryonic stem cells during the development of the embryo with cystic fibrosis, however, other complications such as the uncertainty of whether this technique is safe and the effects it may have on other parts of the genome should be acknowledged.

6.2 Cystic Fibrosis Mutation Diversity

In reality, there are multiple types of CFTR mutations, meaning there isn’t a universal sgRNA to complement all the different types of mutations (Castellani et al., 2008). Therefore, it will be challenging to determine specifically which type of CFTR mutation occurred and thus, difficult to select the correct sgRNA for the corresponding type of mutation. However, researchers are actively developing libraries of sgRNA to compensate for this issue.

6.3 Ethics and Consensuality

Finally, gene editing raises significant ethical concerns and controversy. For example, gene editing is much easier to perform on an embryo, but many people consider it a step too far, believing that individuals should have the choice to decide whether or not they want to take the associated risks (Sadeghi, 2023). As a result, whether the application of gene editing on cystic fibrosis will be fully approved and accepted still remains unknown.

7. Conclusion

In conclusion, with the aim of solving the fundamental problem in the gene as opposed to improving symptoms, CRISPR-Cas9 gene editing is undeniably revolutionary. It has an immense potential in treating genetic diseases such as cystic fibrosis as technology advances. However, certain challenges like off-targeting modification and mutation diversity still remain, therefore it is essential for researchers to find ways to address those issues before implementing this technique for public use.

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