Introduction
The versatile genome editing tool CRISPR/Cas9 has long been the centre of attention for clinical research as it holds great promise: being a rapid and easy way to, theoretically, modify any genomic sequence. CRISPR/Cas9 technology involves the use of an RNA guided mechanism to effectively edit a targeted genome sequence, giving it the potential to become a very important player in the future of medicine. In this essay I will discuss how CRISPR/cas9 technology could significantly impact the future of medicine; through the treatment and possible cure of genetic disorders such as sickle cell disease and beta thalassemia.
CRISPR/cas9 allows targeted disruption of DNA
CRISPR/cas9 has been derived from the prokaryotic immune system and is being used for targeted genome editing. There are 3 types of CRISPR mechanisms (Biolabs, 2018) but here I will be discussing type II as it is the most commonly used. The type II system consists of guide RNA (gRNA) (Li et al., 2018), which is complementary to the target sequence and the cas9 protein: a DNA endonuclease which acts as the molecular scissors. As shown in figure 1: the gRNA acts as a sort of homing device to direct cas9 to the target sequence by complimentary base pairing (Hussain et al., 2018). This target sequence is determined through complimentary base pairing, so you can change the genomic target sequence by changing the sequence in gRNA (Addgene.org, 2018). The target sequence is found 3 base pairs upstream of the protospacer adjacent motif (PAM) (Zhang et al., 2016). The cas9 endonuclease generates a double strand break (DSB) (Ding et al., 2016) which is then repaired by one of two mechanisms: non-homologous end joining (NHEJ) or HDR (homology directed repair) (Platt et al., 2014). NHEJ results in insertions or deletions whereas HDR results in precise sequence substitution. This mechanism can be exploited for targeted and more precise genome editing; when compared with other genome editing techniques.
Superiority over other genome editing techniques
Before the development of CRISPR/cas9 technology, zinc finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENS) were used as the primary genome editing techniques. The most significant difference in these techniques is that CRISPR is based on RNA-DNA interaction whereas ZFNs and TALENS are both based on protein-DNA interaction (Tasan, Jain and Zhao, 2016). Since the introduction of CRISPR/cas9, these techniques have proved to be inferior. Although they have the same function: to create a DSB (Khadempar et al., 2018) CRISPR/cas9 is more cost-effective and not as time consuming. This is because ZFNs and TALENs require the design and generation of a new nuclease (Li et al., 2018) creating a limitation in targeting the desired sequence. The significance, on the future of medicine, of the superiority of CRISPR/cas9 is that it paves the way for faster, more effective gene therapy: compared to what is already available.
Applications in medicine
CRISPR has many applications when it comes to advancements in medicine, however, here I will be focusing on how CRISPR/cas9 is beginning to transform the way in which we treat various blood diseases. CRISPR/cas9 technology is the ideal approach to treating diseases such as sickle cell disease (SCD) and Beta thalassemia as it has the potential to correct the mutations that cause them.
SCD is an autosomal recessive disease (Vinod et al., 2016) caused by a point mutation in the human haemoglobin beta (HBB) gene (Park et al., 2016), this mutation results in a single nucleotide substitution where the hydrophilic glutamic acid at position 6 is converted to a hydrophobic valine (Tasan, Jain and Zhao, 2016): resulting in abnormally shaped red blood cells. Currently, stem cell transplantation is the only viable cure (Park et al., 2016). Thus, the genome editing capabilities of CRISPR could correct this mutation and provide a more promising treatment.
It has been found that gene corrected HBB cells have the potential to differentiate into cells that permanently produce wild-type haemoglobin (Park et al., 2016). They demonstrated that it is possible to correct the mutation with an HDR (homology directed repair) rate of only 30%, meaning that although somewhat successful further research should be carried out before CRISPR/cas9 technology is implemented clinically.
The mutations that cause Beta thalassemia, another blood disorder characterised by mutations in the HBB gene, have also been successfully corrected by CRISPR/cas9 technology. When combined with the piggyBac system, a transposon that transposes between vectors and chromosomes using a ‘cut and paste’ mechanism (Zhao et al., 2016), CRISPR/cas9 efficiently corrected mutations in the HBB gene (Xie et al., 2014).
Research shows that is possible to edit genomes, knocking out the mutations that cause numerous fatal diseases making CRISPR/cas9 technology a key player in the advancement of medicine. However, as with most new discoveries CRISPR comes with limitations that, before being implemented clinically, must be overcome.
Challenges associated with CRISPR/cas9 technology
Despite its clinical potential CRIPSR has certain technical issues that must be addressed, one of these being the off-target effects (Zhang et al., 2015). Large genomes may have DNA sequences that resemble the target sequence, leading to the non-specific cleavage of DNA at a non-target gene (Kang et al., 2017). This non-specific cleavage could lead to unwanted mutations. To combat this, it is necessary that a way to detect off-target sites is found. Another significant challenge faced by researchers is the safe delivery of the CRISPR/cas9 system to cells in the body (Liu et al., 2017).
As well as technical issues CRISPR also has various ethical implications. Possibly the most important of these being the questionable safety of CRISPR treatment, editing the genome could result in irreversible side effects (Brokowski and Adli, 2018) and even death. The ethical debate surrounding CRISPR has two main sides: those who believe research should persist to advance clinical research and those who believe editing the human genome crosses an ethical line (Caplan et al., 2015). Settling this debate depends on further research into CRISPR technology: finding a safe and effective delivery method and a way to minimise off target effects.
Conclusion
Without a doubt, the CRISPR mechanism has many possibilities where the future of medicine is concerned. Its unique gene editing capabilities make it an exciting tool that has the ability to accelerate research in multiple disciplines such as cancer research, drug discovery and disease modelling. Not only this, CRISPR could make it possible to eliminate hereditary diseases and has compelling applications for personalised medicine.
Having said this, despite the potential of this technology improvement is needed before we can dub it the future of medicine. The technology needs to be optimised to prevent off target effects and even once this is done there is no regulation on the use of CRISPR: it could be said that a set of rules needs to be created and implemented worldwide, to prevent misuse of the technology and allow consistent monitoring.
...(download the rest of the essay above)