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CRISPR: What You Need to Know – Sridhar G Kumar

In recent times, with increased media coverage and with the release of movies involving gene editing, one technology involved in this process has been gaining a lot of attention, namely, CRISPR. This technology, although, now widely known for its potential in genetic editing is still not understood by the general populous and exact details of what it is and its current research scope is often ignored for a more far-fetched idea of its future possibilities of creating “designer” or “super” humans and the ethical concerns regarding such applications. In this article, however, we take a closer look at this technology and what it means before we have a closer look at its scope of applications.

Image from Pixabay

CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments from viruses that have previously infected the prokaryote and are used to detect and destroy DNA from similar viruses during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes.

Cas9 (or “CRISPR-associated protein 9”) is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms. This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases.

Diagram of the CRISPR prokaryotic antiviral defense mechanism

The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA. CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.

CRISPR gene editing is a method by which the genomes of living organisms may be edited. It is based on a simplified version of the bacterial CRISPR/Cas (CRISPR-Cas9) antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell’s genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added.

Overview of CRISPR gene editing

While genomic editing in eukaryotic cells has been possible using various methods since the 1980s, the methods employed had proved to be inefficient and impractical to implement on a larger scale. Genomic editing leads to irreversible changes to the gene. Working like genetic scissors, the Cas9 nuclease opens both strands of the targeted sequence of DNA to introduce the modification by one of two methods. Knock-in mutations, facilitated via Homology Directed Repair (HDR), is the traditional pathway of targeted genomic editing approaches. This allows for the introduction of targeted DNA damage and repair. HDR employs the use of similar DNA sequences to drive the repair of the break via the incorporation of exogenous DNA to function as the repair template. This method relies on the periodic and isolated occurrence of DNA damage at the target site in order for a repair to commence. Knock-out mutations caused by Cas9/CRISPR results in the repair of the double-strand break by means of NHEJ (Non-Homologous End Joining). NHEJ can often result in random deletions or insertions at the repair site disrupting or altering gene functionality. Therefore, genomic engineering by CRISPR-Cas9 allows researchers the ability to generate targeted random gene disruption.

Because of this, the precision of genomic editing is a great concern. With the discovery of CRISPR and specifically the Cas9 nuclease molecule, efficient and highly selective editing is now a reality. Cas9 allows for a reliable method of creating a targeted break at a specific location as designated by the crRNA and tracrRna guide strands. Cas9 derived from Streptococcus pyogenes bacteria has facilitated the targeted genomic modification in eukaryotic cells. The ease with which researchers can insert Cas9 and template RNA in order to silence or cause point mutations on specific loci has proved invaluable to the quick and efficient mapping of genomic models and biological processes associated with various genes in a variety of eukaryotes. A newly engineered variant of the Cas9 nuclease has been developed that significantly reduces off-target manipulation. Called spCas9-HF1 (Streptococcus pyogenes Cas9 High Fidelity 1), it has a success rate of modification in vivo of 85% and undetectable off-target manipulations as measured by genome wide break capture and targeted sequencing methods used to measure total genomic changes.

CRISPR-Cas genome editing techniques have many potential applications, including medicine and crop seed enhancement. The use of CRISPR-Cas9-gRNA complex for genome editingwas the AAAS’s choice for breakthrough of the year in 2015.

Arguably, the most important advantages of CRISPR-Cas9 over other genome editing technologies is its simplicity and efficiency. CRISPR technology is very simple, easy to use and cheap unlike the previous gene editing techniques such as Transcription activators-like effective nucleases (TALENS). This technology can be employed to analyze the interaction of genes and relationship between genetic differences and expression (phenotype). It can also be used to knock out gene and replaced it with another gene for diseases therapy

Since it can be applied directly in embryo, CRISPR-Cas9 reduces the time required to modify target genes compared to gene targeting technologies based on the use of embryonic stem (ES) cells. Improved bioinformatics tools to identify the most appropriate sequences to design guide RNAs and optimization of the experimental conditions enabled very robust procedures which guarantee successful introduction of the desired mutation.

The main advantage of using CRISPR-Cas9 in ES cells, compared to the traditional gene targeting approaches, is that Cas9-induced DNA damage increases the frequency of homologous recombination events by many orders of magnitudes. As a consequence, there is no need to identify ES cell clones carrying the modified gene, which streamlines procedures for generating the targeting vector, ES cell screening, and validation. By isolating clonal populations of cells, it is possible to avoid mosaicism and perform in-depth quality control procedures to verify that the modified gene does not carry any passenger mutations.

Due to increase of population and high demand of food worldwide, it is reported as a result of changes of climate and global warming there will be high increase of poverty and food shortage. The emergence of CRISPR-Cas9 has shown greater promise in improving crop yield and preventing crop genetic diseases. This technology can be employed to modify plant genome. The application of this trending technology is increasing rapidly with the aim of developing non-transgenic genome edited plant to avoid adverse changes that may occur as a result of climate change. Scientists have rendered wheat invulnerable to killer fungi like powdery mildew, hinting at engineered staple crops that can feed a population of 9 billion on an ever-warmer planet. CRISPR-Cas9 enables genetic modification of plants to a new level such as modifying them to adapt to changes in climate and improve tolerance to harsh conditions, less diseases and improved crop quality and yield. A group of researchers also worked on modified tomatoes by engineering quantitative gene promoter sequences and this approach led to positive changes in the shape and size of the tomatoes. Another research on rice has shown increase in seed size, number and grain weight.

Using CRISPR researchers have also been working on reversing mutations that cause blindness, stop cancer cells from multiplying, and making cells impervious to the virus that causes AIDS. Bioengineers have also used Crispr to alter the DNA of yeast so that it consumes plant matter and excretes ethanol, promising an end to reliance on petrochemicals.

Another current application of this technology is the introduction of deleterious genes into malaria carrying mosquitoes, one of the ways to tackle malarial diseases. This involves an increased number of fertile female mosquitos to occur with time in the hope to successfully eradicate the disease.

The future for the use of CRISPR technology is unlimited, it cannot be quantified but can only be predicted. According to Doudna et al 2017, CRISPR technology is aimed to help treat cancer, to engineer human beings that have strong bones, are less susceptible to cardiovascular diseases, or to design human with specific traits and for parents to pick baby’s sex, eyes, height and other desirable traits. Scientists have also devoted time to research on anti-ageing, hoping that with this technology, human life expectancy will be over 100 years.

A major misconception regarding the use of CRISPR-Cas9 for genetic editing is the notion that any genetic modifications would in turn affect the genes of their descendants as well. But this is, however, not particularly true. The use of CRISPR to fix genetic disorders and other ailments usually involves the modification of somatic cells and these modifications are limited to the individual. Only changes made to germline cells are inherited to the next generation and these in turn have far reaching consequences.

Currently most applications of CRISPR especially involving humans is somatic editing. However, germline editing even in animals that are not considered particularly essential to the ecosystem must be done with utmost care and in a controlled environment as the ramification of doing otherwise could be disastrous and the damage done irreversible. And this raises the major ethical concern regarding gene editing: how far can we go with tampering with natural evolution before it is considered as too much. Also, what are the traits and aspects that must be considered for editing also is debatable and impossible to reach a common consensus on.

The current scope of CRISPR enables us to envision a future depending on what kind of person we are, either a future with limitless possibilities where we are free of the issues that currently plague the human society or a dystopian future where we have brought in a whole new range of troubles upon ourselves. Without a doubt CRISPR technology is revolutionary, and like all revolutions, it’s perilous. CRISPR could at last allow genetics researchers to conjure everything anyone has ever worried they would — designer babies, invasive mutants, species-specific bioweapons, and a dozen other apocalyptic sci-fi tropes. It brings with it, all-new rules for the practice of research in the life sciences. But no one knows what the rules are — or who will be the first to break them. One can only hope that this technology is used with careful considerations and any germline editing is carried out with extreme care after making detailed analysis of the effects on our ecosystem.


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