Therapeutic Uses of CRISPR-Cas9

Introduction

CRISPR-Cas9 technology has been established as an effective means of gene editing within many different organism forms, resulting in many therapeutic uses. The technology originated in 1987 when a new form of a repeated sequence was found in prokaryotic cells. From 2000-2002 the features of the repeated sequences were found to be made up of collections of short recurring palindromic sequences of 20-40 bp which are divided by unique 20-60 bp sequences. They were found to exist in archaea and bacteria, in 2002 the sequences were termed as (CRISPRs) Clustered Regularly Interspaced Short Palindromic Repeats. In 2007 the CRISPR-Cas technology was confirmed to provide resistance to bacteriophages. In more recent years the molecular, structural and functional characteristics have been discovered of the proteins which are encoded by the Cas gene, which has led to the creation of the CRISPR-Cas9 editing system (Morange, M. J Biosci, 2015). This essay will provide an insight on the CRISPR-Cas9 technology, describing its mechanisms, its application to gene therapy and the therapeutic implications of the technology.

What is CRISPR-Cas9

CRISPR-Cas9 is a technology which allows geneticists and medical researchers to edit parts of the genome, it has the ability to correct faults which are present in the genome. The gene-editing technology can switch on or off genes in organisms or cells simply, rapidly and inexpensively. As well as correcting gene defects, the technology can be used for preventing the spread of disease and improving crops (U.S National Library of medicine, 2020).

How CRISPR-Cas9 works

CRISPR are stretches of DNA, Cas9 is a protein which acts as a molecular scissors which can cut strands of DNA. CRISPR was modified from the characteristics of single-cell microorganisms such as archaea and bacteria, which use CRISPR-derived RNA and Cas9 to stop outbreaks of viruses and other foreign bodies. This is done by Cas9 cutting up and terminating the DNA of such foreign body. These components are made into more complex organisms which therefore permits gene editing. The CRISPR-Cas9 works in the same way in the lab, a small part of the RNA binds to a target sequence of DNA within a gene, it also attaches to the Cas9. Like within bacteria, the RNA which has been modified distinguishes the sequence of DNA, the Cas9 then cuts the DNA at the targeted point (U.S National Library of medicine, 2020).

Applications of CRISPR-Cas9

CRISPR-Cas9 has numerous applications; it has been found to cure mice of genetic disorders by repairing DNA which is defective, human embryos can be similarly modified using the technology and also the treatment of infectious diseases such as HIV and cancer using gene therapy (U.S National Library of medicine, 2020).   

Mechanism of CRISPR-Cas9

When a virus invades a cell, it injects DNA into the cell, the CRISPR technology allows the DNA to be taken out of the virus and inserted in small parts into the chromosomal DNA of the bacterium, the combined parts of viral DNA are inserted at the CRISPR sight. This provides a genetic record of previous infection that allows the host to prevent future invasion from the same invader. Once the DNA has been inserted into the bacterial chromosome the cell will then proceed to make a copy of the RNA, which is an exact replica of the viral DNA. RNA allows interactions with DNA molecules that have a matching sequence.

The RNA from the CRISPR locus bind to Cas9 which is a protein and therefore form a complex which examines all the DNA within the cell. The complex searches for sites that will match the sequences in the RNA which is bound, the sites which are found allows the Cas9 to cut up the viral DNA. The Cas9- RNA complex acts as a scissors which can cut DNA, making double stranded break in the DNA helix. This complex has been discovered as ‘programmable’, meaning it can be programmed to recognise specific DNA sequences and make a cut in the DNA at that certain sight.

This technology was discovered to also apply to genome engineering, allowing cells to make very exact changes to the DNA at the site where the cut has been made. Cells have the ability to detect broken DNA and repair it by either sticking together the ends of the broken DNA with a small change in the sequence at that position, or by joining in a new piece of DNA at the cut sight. Therefore, if double stranded breaks can be introduced at specific sites, cells can also be prompted to repair the cuts by the disruption or incorporation of new genetic information, hence the discovery of genomic engineering. CRISPR is a relatively easy method of genome engineering, once a double stranded break has been made in the DNA it can be repaired and potentially achieve the ability to correct mutations in the cell which cause many life-threatening diseases.          

Application of CRISPR-Cas9 to gene therapy

Gene therapy is a technique which uses genes in the treatment or prevention of disease, it allows healthcare professionals to treat certain disorders by inserting a gene into a patients cell rather than using drugs or surgery. CRISPR-Cas9 has become an easy to use and cost effective method of gene therapy. The CRISPR-Cas9 technology is being widely developed in order to restore disease-causing genes by changing the DNA sequence at its specific location. In recent discovery the CRISR-Cas9 technology has been developed as a potential treatment of HIV1/AIDS by targeting the HIV1 genome in order to stop the HIV1 infection, and allow transcriptional activation of pathogenic viruses for removal. The CRISPR-Cas9 technology has been effectively applied to prevent and reduce HIV1/AIDS in human cells.

CRISPR-Cas9 technology application in HIV1/ AIDS treatment 

The first application of the CRISPR-Cas9 technology in HIV1/AIDS was effectively used to suppress HIV1 expression in Jurkat cell lines which are a preserved line of human T lymphocytes used to study chemokine receptors which are vulnerable to viral entry, predominantly HIV. By targeting the HIV1 LTR (long terminal repeat) the HIV1 provirus was inhibited, preventing further replication and transcription within the cell. Therefore, displaying that CRISPR-Cas9 can remove viral genes from the infected chromosome (Q Xiao, 2019).

CRISPR-Cas9 technology application in Huntington’s Disease

Huntington’s disease causes the degeneration of nerve cells in the brain, the disease has many affects such as uncontrolled movements, psychiatric disorders and the loss of the ability to think. This disease is a genetic disorder, the mutation causes an expansion of the CAG repeat in the gene. An adapted CRISPR-Cas9 approach must be developed for each given Huntington’s disease patient, in order to identify the pairs of PAM sequences that are absent from the regular chromosome but present on the patient’s mutant chromosome. The technology is then used to simultaneously target two PAM sites specific to each patient, without altering the normal allele but eliminating the promotor region and the CAG expansion of the mutant allele. This specific CRISPR-Cas9 approach allows for the inactivation of mutant alleles, and prevent any further mutations in the human genome (Jun Wan Shin, et al. 2016).

CRISPR-Cas9 application to help solve the Food Crisis

The food crisis is currently affecting many countries all around the world, this is because there is not enough land to produce enough food for all of the affected countries, without destroying the Earth’s climate. Scientists have begun research into the production of different foods using the CRISPR technology, foods like jointless tomatoes, fungus-resistant bananas, higher yield corn, soy and wheat have been successfully made. The scientists can look for what genes they must target and which gene edits must be made. Because CRISPR has the ability to cut DNA in many organisms, the exploration of genomes in different exotic plants that grow in extreme environments are also being reviewed. The goal for these scientists is to try evolve traits of these exotic plants like growth in saltwater or the ability to withstand droughts, into the food crops by creating many plants with random mutations to the gene of interest and begin to grow them in climates they are likely to encounter. The main target for the use of CRISPR in food production is to reduce carbon, increase nutrients and produce more food per acre with less inputs (Molteni, 2020).

Therapeutic implications of CRISPR-Cas 9 technology

Although the CRISPR-Cas9 technology has shown many successful and promising results in the treatment of life-threatening diseases, there are some concerning issues that also need to be addressed. These concerns include off-target mutations, gRNA production and CRISPR-Cas9 delivery methods.

Off target mutations 

The leading concern regarding CRISPR-Cas9 gene editing, is off-target mutations. Large genomes usually comprise of numerous DNA sequences. These DNA sequences are identical to the target DNA sequences, CRISPR-Cas9 also cleaves these identical sequences which is the cause of mutations at unwanted sites. These mutations are called off-target mutations, which negatively impact the cell as they may cause transformation or cell death. In order for the CRISPR-Cas9 technology to work in a highly specific manner, the target sites with the least off-target sites should ideally be selected. It is important to control the dose of CRISPR-Cas9 as this is also a potential cause of off-target mutations (Feng Zhang, 2014).

gRNA Production

Guide RNA (gRNA) is an important component of the system, by complimentary base pairing it guides the Cas9 to the target sequence, creating a double strand break. gRNA production is also a concerning issue for CRISPR-Cas9 genome editing, as it too hard to apply RNA polymerase II for gRNA production, as RNA polymerase II is involved in the large-scale posttranscriptional processing of mRNA. Although RNA polymerase III, U3 and U6 promotors are being used to make gRNA, these genes can’t produce the necessary cell-specific and tissue gRNA. The application of U3 and U6 base gRNA production is also due to the scarcity of commercially available RNA polymerase III (V. Rees, 2020).

Delivery Methods

The delivery method used for CRISPR-Cas9 is DNA and RNA injection. Questions have been asked if these delivery methods are efficient, as the method should depend on the types of target cells and also the tissues. Future improvements and studies concerning delivery methods are underway in order to develop a vigorous method to ensure the correct delivery of plasmids expressing Cas9 and gRNA. Tufts University and the Chinese Academy of Science have developed a biodegradable synthetic lipid nanoparticle to deliver the CRISPR editing tools into the cell to alter the cells genetic code, this method resulted in a 90% efficiency in gene editing (Feng Zhang, 2014).

Improving CRISPR-Cas9

Constant developments in the technology imply that there are a lot of further developments to come in order to tackle current challenges. Studies have been completed which show that the conversion of Cas9 to Nickase can aid in the reduction of off-target mutations, whilst keeping the effectiveness of on-target cleavages executed by CRISPR. More research into the Class 1 systems are also being carried out, which may give a greater insight into more therapeutic uses for the CRISPR-Cas9 technology (Rees, 2020).

Conclusion

Evidently, CRISPR has a large number of strong advantages from being discovered as the quickest, cheapest and most precise method of gene editing. This scientific discovery has the potential to treat life-threatening diseases and aid in solving the food crisis. The technology provides potential in the ability to deliver these aspirations, which is why a lot of money is being invested into future research and development of CRISPR. Approximately 795 million people are without sufficient food, as well as 80% of life-threatening genes are because of mutations. These numbers display how important this technology is, and what a big impact it would have on millions of lives if the technology was used for the correct reasons (Molteni, 2020).

Ethical issues are a great concern as treatment and products will be expensive as companies will try make back money which was initially invested. This issue is most alarming for developing countries, as they may not have the sufficient capital to afford the needed benefits of the technology. However, before the technology can be tested in a clinical setting scientists must first reduce its off-target errors. CRISPR has the highest success rate for accurately cutting DNA, therefore because this technology is being viewed as so revolutionary scientists will continue to perfect it so humanity can eventually benefit (Molteni, 2020).

References

• Feng Zhang, Yan Wen, Xiong Guo, 15 September 2014, CRISPR/Cas9 for genome editing: progress, implications and challenges, Human Molecular Genetics, Volume 23, Issue R1, Pages R40–R46, https://doi.org/10.1093/hmg/ddu125
• Jun Wan Shin et al. 15 October 2016,  Human Molecular Genetics, Volume 25, Issue 20, Pages 4566–4576, https://doi.org/10.1093/hmg/ddw286
• Molteni, M. (2020). Crispr Can Help Solve Our Looming Food Crisis—Here's How. [online] Wired. Available at: https://www.wired.com/story/gene-editing-food-climate-change/ [Accessed 26 Jan. 2020].
• Morange, M. (2015). What history tells us XXXVII. CRISPR-Cas: The discovery of an immune system in prokaryotes. Journal of Biosciences, 40(2), pp.221-223.
• Rees, V. (2020). How will CRISPR change and evolve in the future?. [online] Drug Target Review. Available at: https://www.drugtargetreview.com/article/52485/how-will-crispr-evolve-in-the-future/ [Accessed 26 Jan. 2020].
• US National Library of Medicine. (2020). What are genome editing and CRISPR-Cas9. [online] Genetics Home Reference. Available at: https://ghr.nlm.nih.gov/primer/genomicresearch/genomeediting [Accessed 26 Jan. 2020].
• Xiao, Q., Guo, D., & Chen, S. (2019). Application of CRISPR/Cas9-Based Gene Editing in HIV-1/AIDS Therapy. Frontiers in cellular and infection microbiology, 9, 69. doi:10.3389/fcimb.2019.00069