This white paper is an exploration of CRISPR-Cas9 and targeted genome editing technology based on its origin, impact, expert opinion and trends. Factually, genome-editing approaches based on recognition of protein of certain DNA patterns, such as the ones involved in the use of meganucleases, ZFNs, and TALENs have been subjected to numerous clinical trials in readiness for use in individual gene therapy (National Academies of Sciences et al., 2017). Consequently, the recent past has led to establishment of a system guided by RNA. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a technologically inspired name for short, repeated segments of DNA discovered originally within bacteria (National Academies of Sciences et al., 2017). Notably, the segments create the ground for the establishment of a series with short RNA patterns paired with Cas9 (CRISPR associated protein 9, and RNS-directed nuclease), that can be designed to enable DNA editing (National Academies of Sciences et al., 2017). Revealingly, the CRISPR-Cas9 comes with numerous benefits compared to the previous methods used for changing genome and has sparked discussions and debates on the application of genome editing in promoting human health (National Academies of Sciences et al., 2017). Just as TALENS, ZFNS, and meganucleases, CRISPR-Cas9 technology has the capacity to form double-stranded breaks within DNA and the cells’ own DNA repair mechanisms to enable ideal genome changes. In fact, compared with other methods, CRISPR-Cas9 is a cheap and easy method to enable the required edits within the genome.
Section 1 - Overview of CRISPR-Cas9: the origins of CRISPR Cas-9, what it does and how it works
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Reports indicate that, Clusters of Regularly Interspaced Short Palindromic Repeats (CRISPR) were for the first time seen within bacteria in 80s, and in the mid 90s, within genomes of various archeal lineages (Barrangou & van der Oost, 2012). According to biological experts, their notable broad distribution was evident that they are likely to play a fundamental cellular responsibility and they might contribute towards the adaptation to extreme environments (Barrangou & van der Oost, 2012). Evidently, their repeat spacers were unique both in sequence and in structure, compared to other systems, and there lacked precedents for identifying their functions (Barrangou & van der Oost, 2012). The first evidence showing their functionality emanated from reports on haloarchae, where defects of growth were seen within cells changed using artificial plasmids. In retrospect, CRISPR-enabled autoimmunity had the ability to explain the unique phenotypes, and specifically chromosome loss (Barrangou & van der Oost, 2012). It was followed by important development, identifying CRISPR-associated (cas) genes, which showed that repeat-spacer patterns were related to CRISPR-Case protein systems (Barrangou & van der Oost, 2012). Based on sequence discoveries and studies, it was revealed that spacers within the DNA were not appropriate and it led to the assertion that the system lead to immunity against the foreign DNA (Barrangou & van der Oost, 2012). As a follow-up, experiments to support this hypothesis were carried out and it showed that, CRISPR-Cas systems contained a rare form of genetic problem. Factually, experts in the field of genetics have overtime classified CRISPR systems into three major types I, II, III, with few subtypes different in formation details (Barrangou & van der Oost, 2012). Besides influencing other cellular pathways, the gene approach employed by CRISPR systems has the ability of functioning as a defense system (Barrangou & van der Oost, 2012). The afore-mentioned is supported by research on the biochemical and genetic characterization of Cas proteins and on scientific activities of different CRISPR-Cas systems.
Following the discovery of CRISPR-Cas, it was attributed to various functions. The revelation that E. coli designs occurred within the intergenic regions led to the assertion that they contributed in the modulation of gene expression (Barrangou & van der Oost, 2012). In the same vein, speculations were evident that DR patterns of mycobacterium were likely to influence expression within the neighboring genes by generating binding sites for regulatory proteins. Additionally, CRISPR-Cas has been mentioned in relation to chromosomal patterns for E. coli and other bacteria (Barrangou & van der Oost, 2012). Besides immunity, recent studies have come up, which seem to associate the CRISPR-Case with numerous functions inclusive of DNA-repair in E. coli.
Experts observe that, CRISPR-Cas9 has come up as a new technological device for DNA using single-guide RNA (sgRNA), making it possible to have genome editing (Barrangou & van der Oost, 2012). The single RNA protein CRISPR emanates from a natural microbial adaptive immune system that utilizes RNA-enabled nuclease to remain compatible to the foreign DNA elements (Yamamoto, 2015). Notably, the CRISPR system has been proven by experts as an incredibly valuable asset for site-specific genome engineering (Barrangou & van der Oost, 2012). For instance, in human cells, the improved Cas9 version known as dCas9 was established for RNA-dependent protein (Yamamoto, 2015). Reportedly, dCas9 is capable of being modulated to enable different protein effectors to DNA in an improved manner to activate a CRISPR gene.
In its context, CRISPR-Cas is applicable in genome editing. While applying it for that purpose, only two elements are useful namely the chimeric guide RNA (gRNA), and the Cas9 protein that has nuclease activity (Yamamoto, 2015). Factually, the targeting specificity is majorly dependent on gRNA sequence, but Cas9 also needs a few particular bases called the Protospace Adjacent Motif (PAM). The sequences of PAM tend to vary within species. For instance, Streptococcus pyogenes Cas9 (SpCas9) requires 5’-NGG-3’ (Yamamoto, 2015). Currently, SpCas9 is applied mostly in genetic engineering. In addition, the SpCas9-gRNA complex is known for selecting out the PAM pattern within genome, and unwinding double strand DNA to form DNA-RNA pairing base, in a directional sequence (Yamamoto, 2015). Reportedly, gRNA structure is yet another significant factor within CRISPR/Cas9-based genome editing. Although it is possible for tracrRNA and crRNA to transcribe separately just as in the natural CRISPR/Cas, a gRNA design is simple and associated with high activity.
Revealingly, genome editing through CRISPR-Cas9 technology works in many ways. One of the major ways it works is by enabling basic science laboratory research. Evidently, it has become useful to scientists in research regarding tissues and cells, which is significant in the field of biomedical science. It has helped to advance the understanding of processes that control disease progression and development, ideally making it possible to control disease development and progression. In the same vein, it makes it possible to come up with better inventions for the individuals affected. CRISPR-Cas9 technology has been vital in the search for germline cells and helpful in advancement areas of fertility treatment and regenerative medicine. Human fertility and development can equally be understood using same technology.
In clinical uses, CRISPR-Cas9 technology has been vital in altering somatic cells for purposes of preventing diseases in patients with advanced cancer. It is used on patients who have been unable to respond to conventional medical interventions such as radiation and chemotherapy. In such cases, genome editing through CRISPR-Cas9 becomes vital in programming the immunity of patients with cancer. Notably, somatic genome-editing therapies using CRISPR-Cas9 is applicable in clinical practices for numerous ways. The technology can be used to remove sections of cells such as in bone marrow from the body of an individual, enabling certain changes in genes and reversing the cells to same person. Markedly, genome editing can be performed directly into the body or outside the body. However, experts’ advice that in some cases, the tools introduced within the body may fail to identify the target gene appropriately.
Section 2 - Impact of CRISPR-Cas9: current ethical considerations associated with CRISPR-Cas 9 and any direct environmental issues associated with CRISPR-Cas9
Numerous ethical, environmental, social concerns regarding gene therapy provide viable ground for consideration of major issues associated with genome editing. With proper and careful oversight, research on gene therapy has enjoyed and exhibited huge support for a wide range of stakeholder groups. Even so, considering that CRISPR-Cas9 has made genome editing abundantly precise and efficient, they have led to numerous applications that continues to be seen as largely theoretical. One of the perfect examples is the application of editing to protect humans from genetically inherited ailments. For the mere fact that genome editing has only began to change from ordinary research to application within clinical settings, this becomes the ideal time to sample its use on humans and put into consideration appropriate ways to advance and apply governance on the scientific discoveries. The rate at which science is advancing has sparked high levels of enthusiasm among the industry, scientists, various health organizations and patients that see the benefits that come with these advances. Among the policy makers, concerns have been raised regarding whether reliable systems are available to govern technologies and whether ethical values will be seen in the way genome editing is being applied in the practice.
Studies after studies have delved in reviewing ethical issues touching on CRISPR-Cas9 technology in relation to genome editing. Its magical ability to allow DNA editing within living cells and the fact that no trace is left has sparked ethical debates among scientists and other genetic experts (Rodriguez, 2017). Ethically, there exists a strong feeling that CRISPR-Cas9 can pose a risk of inducing and causing mutations. Just as is common with genome editing, the application of CRISPR-Cas9 is likely to influence off-target mutations (Rodriguez, 2017). The afore-mentioned is likely to happen whenever CRISPR-Cas9 targets the genes on a repeated basis at various stages of embryo development (Rodriguez, 2017). According to experts in genetic engineering, such mutations are destructive and can end up causing death of the cell (Rodriguez, 2017). Although efforts have been made to minimize the unintended mutations, experts still feel that any CRISPR-Cas9 engineering conducted within an organism need to undergo testing.
Currently, debates on regulation in regards to CRISPR-Cas9 have been on the rise, especially due to its efficiency. Genetic engineers feel that, the absence of an effective and global body handling the application of CRISPR-Cas9 poses an ethical challenge bordering quality. Currently, the bodies such as Food and Drug Administration (FDA), National Institutes of Health (NIH), and Environmental Protection Agency (EPA), have no ability to offer adequate monitoring and control capacity (Rodriguez, 2017). Although the NIH has come up with guidelines governing virulence, pathogenicity, environmental and communicability stability in relation to CRISPR-Cas9, there lacks funding towards the highlighted procedures (Rodriguez, 2017). While sections of biotechnology companies ensure all the procedures outlined are followed, some ignore the procedure, and end up compromising quality (Rodriguez, 2017). Stakeholders in the field of genetics should push for sufficient and reliable mechanisms of controlling the application of CRISPR-Cas9. Questionably, a serious player such as the United States is not part of the United Nations Convention on Biological Diversity.
Indeed, the tendency of placing animals within the confines of a laboratory for gene editing processes has raised ethical concerns. Animals are subjected to a wide range of risks for the sake of CRISPR-Cas9 processes (Rodriguez, 2017). Reports indicate that, some off target mutations have led to fetal abnormalities, and in some cases, genes have lost function. Experts have been pushing for use of non-human animals with CRISPR-Cas9 processes, unless adequate justification is provided (Rodriguez, 2017). Use of animals in research has to be justified whether the process aims at understanding human diseases and other significant biological processes (Rodriguez, 2017). Importantly, experts have to put into consideration whether other alternatives exists to avoid subjecting animals to unnecessary suffering.
From an environmental perspective, it is obvious that CRISPR-Cas9 has impact on the environment, bearing in mind that it is heavily reliant on technology and science. It was discovered with an intention of helping to end spread of ailments (Patra & Andrew, 2015). Although the CRISPR-Cas9 technology comes with numerous benefits, numerous environmental questions have emanated from its use (Patra & Andrew, 2015). The new organisms established through it pose an ecological challenge. However, experts in this field have warned that to some extent, it is difficult to determine the impact of genetically engineered elements on the environment (Patra & Andrew, 2015). Possibly, genetically engineered species have the ability of triggering an imbalance within the ecology. Unknown result of CRISPR-Cas9 is likely to lead to several issues (Patra & Andrew, 2015). For instance, in the event scientists make a mistake in the process of engineering virus or bacteria genetics, a stronger type is likely to be produced (Patra & Andrew, 2015). Consequently, a serious pandemic might be the end result. The impact might be evident on humans and may even result to death.
Notably, CRISPR-Cas9 has been associated with causing ecological disequilibrium. Studies have revealed that, organisms modified through CRISPR-Cas9 are likely to trigger disequilibrium in the ecosystem, considering they might outdo the immediate wild species (Rodriguez, 2017). Whenever the gene nucleus of Ca9 is edited, it might end up generating a gene that is likely to spread a given trait in an entire population, causing an ecological disequilibrium (Rodriguez, 2017). For instance, in 2015, Drosophila was created, and it had a capacity of causing mutation in over 90% of offspring in two generations (Rodriguez, 2017). The Cas9 nucleases were integrated to produce a mutagenic chain reaction. The mutation caused by CRISPR is copied on a chromosome related to its partner to ensure the subsequent generations will inherit part of the edited genome (Rodriguez, 2017). Upon being released outside a laboratory, the traits are spread across the generation. Revealingly, CRISPR-Cas9 technology is capable of eradicating invasive species and disease micro-organisms by eliminating the organisms of causing diseases (Rodriguez, 2017). Notably, experts warn the process might have some unseen secondary impacts. For instance, by editing the Aedes aegypti female mosquito, the process will help minimize its chances of transmitting dengue disease and the process may reduce their offspring’s lifetime (Rodriguez, 2017). However, sections of researchers have asserted that, through CRISPR-Cas9 technology, a gene can be created with the ability to minimize malaria transmission without necessarily destroying the mosquito population.
Section 3 - Perspectives and Opinions: provide alternative perspectives from experts on CRISPR-Cas9 and what political implications and influence does CRISPR-Cas9 pose
Genome editing has gained considerable attention since the introduction of CRISPR/Cas9 technology in 2012. Notably, medicine has undergone radical shifts through introduction of disruptive technologies that allow doctors to approach diseases in advanced ways. CRISPR/Cas9 allows for corrections in DNA and eliminating adverse conditions before they materialize (Rodriguez et al., 2019). A recent example is work conducted by He Jiankui, a Chinese researcher, who edited the genomes of two baby embryos to remove the CCR5 gene, which is associated with HIV/AIDs infections (Bergman, 2019). Jiankui disregarded bioethics and conducted germline editing which is less conventional compared to the somatic gene therapy commonly used by researchers (Bergman, 2019). Somatic gene editing only affects the target genes and some of the cells while germline editing affects all cells. According to Bergman (2019), germline affects eggs and sperms meaning that it can be carried on to offspring. Scientists around the world contend that such situations are leading to multiple perspectives in the application of CRISPR/Cas9 in modern medicine.
Altering the function of a gene can be achieved by inactivating it through homologous recombination or blocking the messenger RNA (Rodriguez et al., 2019). Williams and Warman (2017) indicate that microorganisms have developed new methods of defending against pathogens. CRISPR/Cas9 takes advantage of one of these mechanisms, which is the biological process through which microorganisms target and disable the DNA of a pathogen (Rodriguez et al., 2019). Since 2012, CRISPR/Cas9 technology has advanced restriction endonucleases offering treatment for a wide range of illnesses including cancer, HIV and muscular dystrophy (Isa et al., 2020). Further, the technology is preferred due to its higher targeting efficacy (Isa et al., 2020). The CRISPR/Cas9 system relies on prokaryotes to recognize and store snippets of viral DNA (Williams & Warman, 2017). Prokaryotes generate a self-defense mechanism whenever a foreign pathogen is detected. CRISPR/Cas9 requires about9 proteins and 2 RNAs, however, scientists have been able to reduce the effector arm to two components (Williams & Warman, 2017). The first is the enzyme Cas9, which cuts the DNA strand at the desired point. The second component is the guide RNA that is 20 nucleotides long and designed to bind only to the targeted sequence (Williams & Warman, 2017). In that sense, CRISPR/Cas9 is highly effective due to the nucleotide-based activity.
CRISPR/Cas9 uses germline gene editing to carry out its gene blocking mechanisms. Scientists have debated the ethical and scientific considerations in using both germline and somatic gene editing. Isa et al. (2020) assert that germline gene editing is less complex compared to somatic because it does not require mitochondrial replacement therapy and cell addition. Additionally, CRISPR-Cas9 is cost effective because it is guided by a strand of RNA rather than protein (Crudele & Chamberlain, 2018). Scientists edit the existing genes to prevent embryos from inheriting a genetic disorder. Only healthy embryos are transferred to the womb meaning that parents no longer have to consider abortion for a child that inherits a gene disorder (Isa et al., 2020). Regardless, gene changes made through germline editing on the human genome are inheritable by future generations. According to Crudele and Chamberlain (2018), direct editing of cells uses a viral-directed vector to transfer the edited Cas9 gene leading to long-term expression of the change in the genetic composition. This change may pose a threat to patients lacking anti-Cas9 memory T cells (Crudele & Chamberlain, 2018). It could lead to inflammation due to development of anti-Cas9 CTLs (Crudele & Chamberlain, 2018).
Recent developments have indicated that CRISPR/Cas9 could be used to target multiple genomic loci. Williams and Warman (2017) indicate that scientists achieved this by discovering new protein complexes with novel DNA and RNA compositions. Also, scientists have modified the Cas9 protein to bind only a single DNA strand or target a specific nucleotide (Williams & Warman, 2017). This has significantly improved homology-directed DNA repair (HDR), which makes it possible to create alleles that contain specific modifications (Williams & Warman, 2017). Furthermore, religious beliefs are among alternative perspectives to issues of gene editing. Isa et al. (2020) contend that Islamic perspectives are against germline gene editing due to its impacts on human dignity and tampering with God’s creation. The American Society for Gene and Cell Therapy (ASGCT) has proposed banning all human germline gene editing until all technical and ethical issues are solved (Isa et al., 2020). Additionally, a summit of scholars held in 2015 emphasized the need to suspend all germline editing until safety and efficacy issues are established (Isa et al., 2020).
Dr. Jiankui’s announcements on gene editing sparked a lot of response to the issue of germline gene editing. Different parties are calling for stringent regulations to control how scientists develop this technology to avoid bioethical concerns (Morrison & Saille, 2019). In December 2015, Congressman and physicist Bill Foster, opened the US National Academics of Sciences, Engineering and Medicine summit where he reiterated the importance of gaining public importance on CRISPR/Cas9 gene-editing technology (Morrison & Saille, 2019). Morrison and Saille (2019) indicate that some jurisdictions such as the UK strictly control human gene editing through the Human Fertilization and Embryology Authority (HFEA). However, lack of an international level of standards has led to disparities between jurisdictions resulting in difficulties regulating unproven medical interventions as seen in the case of He Jiankui.
Conversely, Bergman (2019) indicates that regulations on CRISPR/Cas9 gene-editing technology should consider the concepts of governance and self-governance. Scientists make their decisions based on research conducted within the scientific community using peer reviews and public censure (Bergman, 2019). Dr. Jiankui’s case should not affect other researchers who are adhering to bioethical standards. Public policy that fails to consider the progress of scientists is detrimental (Bergman, 2019). There should be close discussions between policymakers and researchers to create an international consensus since laws and regulations are rarely effective in handling a transnational problem (Crudele & Chamberlain, 2018). Ultimately, debates should focus on bringing more sensitivity to alternative perspectives to create an equitable approach to gene editing.
Section 4 - Trends: current PoC (Proof of Concepts) for alternative application of CRISPR-Cas9 and gaps in the current application of CRISPR-Cas9, or opportunities to drive towards new market
According to National Institutes of Health (NIH) (2015), international researchers on CRISPR-Cas9 have established three new naturally-enabled systems that can be applied in genome editing. The discovery is a huge milestone in the field of biomedical research. the systems have same features with Cpf1 and Cas9, a recently discovered enzyme, but it has rare characteristics that can be utilized in genome editing applications (National Institutes of Health, 2015). The researchers used a bioinformatics approach in discovering the new proteins namely C2c1, C2c2, and C2c3 resulting into numerous series that can be used in place of CRISPR-Cas9. Scientists believe that the new mechanisms are without a doubt going to attract the attention of scientists (National Institutes of Health, 2015).
One of the most recent alternative to CRISPR-Cas9 is the Cas-CLOVER. Factually, CRISPR-Cas9 has shown the world it has the ability to facilitate gene editing. Mostly, it is suitable in non-commercial settings, and experts have been questioning whether it is suitable for application within commercial settings (Xianghong et al. 2018). At the same time, CRISPR-Cas9 utilizes a single RNA guide, which users have identified as capable of creating off target mutations. In contrast, the newly discovered Cas-CLOVER has been associated with clean issued patents and is hardly associated with off-target activities (Xianghong et al. 2018). The system has been identified as capable of maintaining efficiency, and is easier to use compared to CRISPR-Cas9 (Xianghong et al. 2018). The technology has been licensed by HERA. Though almost similar to CRISPR-Cas9, it uses a rare nuclease protein known as Clo51, covered by patents different from the CRISPR-Cas9 system (Xianghong et al. 2018). The Cas-CLOVER is a fused protein, and contains a nuclease-enabled Cas9 protein, connected to the Clo51 endonuclease (Xianghong et al. 2018). It has impeccable specificity considering that it utilizes a double-guide RNA. When compared to CRISPR-Cas9, the Cas-CLOVER displays high efficiency in disrupting the targeted gene (Xianghong et al. 2018). The Cas-CLOVER strategy of using tow RNAs leads to highly reliable genome editing tool.
Revealingly, CRISPR-Cas9 has demonstrated great ability in the elimination of some deadly disorders and diseases among them the Huntington’s disease. Even so, owing to some nonspecific genome effects, its future development has been placed under threat (Valero, 2018). In that regard, researchers at the University of Texas, Austin, have proposed the alternative of CRISPR-Cas9 with a protein known as Cas12a, owing to its ability of solving the limitations displayed by CRISPR-Cas9 (Valero, 2018). According to the experts, the Cas12a is efficient than CRISPR-Cas9 because it has a unique coupling. Cas-9 utilizes the first letters in genomic destination, and ignores the rest of the positions (Valero, 2018). The approach ends up causing the discrepancy in the formation of the DNA structure and the erroneous editing can cause harm to the body (Valero, 2018). In contrast, Cas12a operates in an effective way that ensures none of the positions is missed, ensuring no errors capable of arising (Valero, 2018). The afore-mentioned facts have been showcased by University of Texas researchers.
Owing to its toxic nature, CRISPR-Cas9 has sparked a concern among scientists. Consequently, scientists have been compelled by the faults to come up with a method aimed at improving it (Valero, 2018). A group of scientists from the National Institute of Standards and Technology and the Stanford University made a revelation that they had managed to discover a new editing system known as MAGESTIC, designed with the ability to improve the process of DNA repairing (Valero, 2018). In 2018, another report was made by Poland scientists that they had managed to come up an improved type of CRISPR by using the Cas9 variant, capable of dividing a DNA sequence into two (Valero, 2018). The process is capable of reducing the risks of generating the nonspecific deletions of the genetic elements.
In terms of gap, the CRISPR-Cas9 though a powerful and reliable tool for gene editing still needs to be improved (Campbell & Hyde, 2017). Notably, the Cas9 has nucleus that can be programmed to enable it use a short designed RNA. The fact that CRISPR-Cas9 can be programmed with ease makes it to be adopted and used in a wide range of research areas and applications such as in gene function and in treating genetic disorders (Campbell & Hyde, 2017). However, the system has the tendency to go off-target and aim at other sites. This challenge has remained a concern for scientists considering that off target leads to uncertainty in scientific discoveries (Campbell & Hyde, 2017). In that sense, it is advised that CRISPR-Cas9 is used with a lot of care, especially when it comes to therapeutic applications (Campbell & Hyde, 2017). For that reason, considerable effort is needed to help discover remedy for off target activities in CRISPR-Cas9.
Currently, CRISPR-Cas9 has presented great opportunities and has played a huge role in improving the quality of life to many suffering patients (Valero, 2018). In that respect, the positive results have opened opportunities for large institutions such as the Harvard, Cambridge, the Broad Institute of MIT and the USA in establishing Zhang Lab institute of research committed to studying genomics in the biomedical field (Valero, 2018). The afore-mentioned parties have seen a viable opportunity to commit resources for recruiting new talent, empowering genetic science and offering support in research. Recently, researchers from Zhang lab published their findings in Science regarding a Cas9 variant known as SpCas9-NG (Valero, 2018). In that sense, CRISPR-Cas9 is promising for the future generations as there is still a room for improving it.
According to research by Campbell & Hyde (2017), one of the prestigious opportunities in CRISPR-Cas9 that needs to be driven in the new market is retinal degeneration research. Notably, researchers began the process of finding out how they can use the zebra fish to restore sight to individuals who are visually impaired (Campbell & Hyde, 2017). Researchers reveal that, applying CRISPR-Cas9 towards the retinal regeneration will help in understanding the process of repairing retinal and restoring vision (Campbell and Hyde, 2017). Apparently, mammals and humans lack the ability to regenerate damaged retina, but in the case of zebra fish, it can reproduce the retina after loss (Campbell & Hyde, 2017). Whenever human beings experience damaged retina, they do not get cure since it contains post-mitotic neurons. In the past decades, experts have committed effort and resources trying to find treatment for loss of vision such as gene therapy, prosthetics, and transplant among others (Campbell & Hyde, 2017). However, the afore-listed methods pose a huge burden to the caregivers and the affected persons. Regenerative approach through CRISPR-Cas9 seeks to restore and repair the damaged retina. Reports indicate that, zebra fish cone resembles that of human beings both in circuitry and anatomy (Campbell & Hyde, 2017). It makes it the most appropriate for studying by genetic engineers. In that respect, CRISPR-Cas9 genome editing has most recently opened an opportunity for experts to find the appropriate targeted mutations. As it stands, numerous toolsets have been established in an attempt to find protein function and gene expression (Campbell & Hyde, 2017). Additionally, zebra fish genome is extensive and databases are available, which contains zebra fish expression patterns, transgenic and strains (Campbell & Hyde, 2017). Undoubtedly, CRISPR-Cas9 genome editing will be the only available hope for humans to restore retina emanating from disease or trauma (Campbell & Hyde, 2017).
Conclusion
Having seen the role CRISPR-Cas9 has played in genome editing, this white paper has examined its potential role in curing diseases and disrupting the mutations or in helping to insert new genes for protective functions. The technology is significant in altering the way genes in an organism work, through substituting or adding DNA sections. Since its origin in the 80s and 90s, the CRISPR-Cas9 has come a long way to improve living things in overcoming some inherited genetic problems and in modulation of gene expression. However, the CRISPR-Cas9 has sparked numerous ethical and environmental issues. Scientists have questioned the current regulations governing its use, and the fact that it has the ability of causing ecological imbalance. To improve on its reliability, experts advise that the off-target activity needs to be rectified in order to get desired results, especially in clinical applications. CRISPR-Cas9 has opened opportunities in the field of genetic engineering and new trends have equally emerged, as experts target to introduce suitable alternatives for genome editing. For instance, Cas-CLOVER has been identified as a perfect alternative of CRISPR-Cas9 owing to its unique trait of utilizing a double-guide RNA, thus being extremely efficient. In terms of opportunity, scientists and researchers continue to explore various possibilities of ensuring that the zebra fish retina is used in restoring sight to individuals with visual impairment problems.
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