A 2014 MIT Technology Review article by A Regalado described CRISPR as “the biggest biotech discovery of the century.” This was not an overstretch. CRISPR gene editing is a genetic engineering technique based on the bacterial CRISPR-Cas9 antiviral defense system. It is fundamentally a “search and replace” function for DNA that could be used to disable genes or change their functions by deleting or replacing targeted DNA letters or predefined sequences.
Nevertheless, studies related to the scientific and therapeutic applications of CRISPR range from producing genetically modified organisms to reengineering the body to cure genetic disorders and other illnesses. Furthermore, several studies suggest the possibility of rendering the human immunodeficiency virus unable to replicate inside an infected immune cell.
This article provides a simplified and concise discussion explaining what is CRISPR and how CRISPR/Cas system gene editing works.
Explaining the Basics of CRISPR Gene Editing
Defining and Understanding Gene Editing
Genome editing or gene editing with engineered nucleases is a specific genetic engineering process that involves using engineered nucleases or molecular scissors to insert, delete, or replace DNA or specific DNA letters and sequences. Within the realm of medicine, it is tantamount to reengineering the body to correct genetic disorders or create a response to a particular illness by inducing targeted mutations.
Several gene editing methods have been developed over the last decade. One of these involves using. One method involves using artificial nucleases such as zinc finger nucleases or ZFNs and transcription activator like-effector nucleases or TALENs capable of inducing targeted mutation by inserting, deleting, or editing DNA.
The applications of ZFNs and TALENs have produced results. These include targeted gene modification in plants to increase their resilience and improve yield, as well as gene therapy to manage or treat several disorders.
Despite its current and potential applications, Koyanagi et al. reiterated that both ZFNs and TALENs remain somewhat difficult and time-consuming to design and develop. However, in 2013, researchers Jennifer Doudna and Emmanuelle Charpentier discovered another gene editing method that involved using the CRISPR system. When compared to ZFNs and TALENs, it is simpler to design and develop.
Other researchers have explored further the properties and application of CRISPR. The emerging discoveries have created high hopes for using CRISPR gene editing or the more specific CRISPR/Cas system gene editing in different medical and biotechnological applications.
History of CRISPR Discovery and Research
It was in 1987 when Yoshizumi Ishino and her colleagues at Osaka University in Japan published a study detailing the sequence of a gene called iap found in the gut of E. coli. To better understand how this gene worked, they also sequenced some of the DNA surrounding it. They found that near the iap gene are a clustered repeats or identical segments of DNA. But the function of these repeats was not clear at this time.
Other independent studies also reported these clustered repeats present in other species of bacteria and archaea. These findings reported that each repeat sequence was separated from the other by DNA spacers. These spacers had a unique sequence, unlike the repeat sequences.
A 2002 study by researchers Ruud Jansen et al. collectively called the repeat sequences and the DNA spacers between them as clustered regularly interspaced short palindromic repeats or CRISPR. They also explained that a collection of genes accompanied the CRISPR sequences. They called these genes CRISPR-associated genes or Cas genes.
What was interesting about these Cas genes was that they encoded putative nuclease or helicase protein—or enzymes that could cut or unwind DNA. Jansen et al were unable to explain why these genes did so. Furthermore, researchers could not explain why these Cas genes always sat next to the CRISPR sequence.
In 2005, three separate studies from separate researchers noticed something odd about CRISPR spacers: they resembled viral DNA and extrachromosomal DNA such as plasmids. The three groups of researchers drew several conclusions and suggestions from their studies.
The study of C. Pourcel, G. Salvignol, and G. Vergnaud concluded that the CRISPR structure provides a new and robust identification tool for evolutionary studies. Another study by Elena Soria et al. concluded that the extrachromosomal elements in CRISPR fail to infect the specific spacer-carrier strain, thereby implying a relationship between CRISPR and immunity against targeted DNA.
Another study by A. Bolotin et al. also suggested that the spacer elements in CRISPR represent traces of past invasions by extrachromosomal elements. They further hypothesized that CRISPR provides cells immunity against phage infection and foreign DNA expression by coding an anti-sense RNA.
The Function of the CRISPR/Cas System
Further studies about CRISPR confirmed previous hypotheses. As it turned out, CRISPR works together with Cas and crRNA to form the CRISPR/Cas system. This complex is a naturally occurring defense mechanism found in a wide range of bacteria and archaea. J. A. Doudna and her team described CRISPR as essential components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea.
Remember that CRISPR represents the segments of bacterial or archaea DNA containing a short repetition of base sequences. Between the repetitions is a short segment of spacer DNA or intergenic spacer acquired from previous exposure to plasmids or viruses. The entire CRISPR/Cas system is a complex composed of CRISPR RNAs or crRNAs and CRISPR-associated proteins or Cas proteins. This complex works by degrading the complementary sequences of invading viral or plasmid DNA.
Doudna et al. explained that the CRISPR-mediated immune systems depend on small RNAs for sequence-specific detection and silencing of foreign genetic elements. The systems form part of the greater immune response of prokaryotic species. To better illustrate, whenever a plasmid or virus invades a microbe, the microbial cell grabs some genetic material from the invader, cuts open its DNA, and inserts the plasmid or viral genetic material into a spacer.
The result of this foreign DNA insertion is the CRISPR/Cas system. This system or complex represents the encounter or exposure to plasmid and viral genetic materials. As an adaptive immune system, the microbe uses the obtained foreign genetic material to turn Cas enzymes into a targeted immune response mechanism alongside RNA targeting. This works by copying the foreign genetic material from each spacer and turning it into an RNA molecule or crRNA molecule. The Cas enzyme then integrates this crRNA molecule.
Whenever the microbe reencounters a similar plasmid or virus, the crRNA recognizes and latches on the plasmid or viral DNA sequence while the Cas enzyme cuts the plasmid or viral DNA into two to prevent it from replicating.
In other words, the CRISPR system is a prokaryotic adaptive immune system that confers resistance to foreign genetic elements such as plasmids and viruses through RNA-guided genetic silencing. To a certain extent, the entire CRISPR array also represents the history of encounters with invading genetic elements.
Application of CRISPR Gene Editing Tool
The properties and mechanism of the CRISPR/Cas system make it a probable genome editing tool—specifically for inserting, deleting, or replacing DNA. According to an MIT Technology Review article, American biochemist and molecular biologist Jennifer Doudna and French molecular biologist and geneticist Emmanuelle Charpentier have been largely credited for introducing the CRISPR/Cas system as a novel gene editing tool.
Both Doudna and Charpentier collaborated to explore and understand the molecular mechanism of a particular CRISPR/Cas system that uses the specific Cas protein called Cas9 found in the Streptococcus bacteria—the bacteria commonly responsible for strep throat infection or sore throat.
Their study demonstrated that Cas9 could be used to make cuts in DNA sequences as desired. Take note that this demonstration involved combining Cas9 with synthetic or lab-developed guide RNA molecules. The RNA molecule could be designed to match a sequence of DNA researchers are targeting to edit.
Nonetheless, Doudna and Charpentier concluded that there is a high potential for exploiting the application of CRISPR/Cas9 system as an RNA-programmable gene editing tool. This means that researchers can design a CRISPR/Cas9 system that can work in any organism.
There are currently other researchers and startup companies working to understand and exploit CRISPR gene editing and CRISPR/Cas system further. Other studies have demonstrated an application centered on activating or silencing genes. The CRISPR/Cas system opens the possibility for treating genetic disorders and even dreaded diseases such as cancers.
It is worth mentioning that the CRISPR/Cas system provides a simple tool for mimicking diseases or demonstrating what happens when a gene is knocked down or mutated.
Other studies showed the use of this gene editing tool to rid cells of infections, while some studies have demonstrated using the CRISPR/Cas system at the germline level to create species with predefined traits by targeting specific genes.
The possibilities from, as well as specific applications of CRISPR gene editing, are starting to emerge as researchers and even the business community have been channeling their resources to perfect or discover novel applications in the fields of medicine and biotechnology.
FURTHER READINGS AND REFERENCES
- Bolotin, A., Quinquis, B., Sorokin, A., and Ehrlich, S. D. 2005. “Clustered Regularly Interspaced Short Palindrome Repeats (CRISPRs) Have Spacers of Extrachromosomal Origin.” Microbiology. 151(8): 2551-2561. DOI: 1099/mic.0.28048-0
- Regalado, A. 2014. “Who Owns the Biggest Biotech Discovery of the Century?” MIT Technology Review. Available online
- Ebina, H., Misawa, N., Kanemura, Y., and Koyanagi, Y. 2013. “Harnessing the CRISPR/Cas9 System to Disrupt Latent HIV-1 Provirus.” Scientific Reports. 3(1). DOI: 1038/srep02510
- Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., and Nakata, A. 1987. Nucleotide Sequence of the iap Gene, Responsible for Alkaline Phosphatase Isozyme Conversion in Escherichia coli, and Identification of the Gene Product.” Journal of Bacteriology. 169(12): 5429-5433. DOI: 1128/jb.169.12.5429-5433.1987
- Jansen, R., Embden, Jan. D. A. van, Gaastra, W., and Schouls, L. M. 2002. “Identification of Genes that are Associated with DNA Repeats in Prokaryotes.” Molecular Microbiology. 43(6): 1565-1575. DOI: 1046/j.1365-2958.2002.02839.x
- Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and Charpentier, E. 2012. “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity.” Science. 337(6096). 816-821. DOI: 1126/science.1225829
- Mojica, F.J., Díez-Villaseñor, C., García-Martínez, J., and Soria. E. 2005. “Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements.” Journal of Molecular Evolution. 60(2): 174-192. DOI: 1007/s00239-004-0046-3
- Pourcel, C., Salvignol, G., and Vergnaud, G. 2005. “CRISPR Elements in Yersinia Pestis Acquire New Repeats by Preferential Uptake of Bacteriophage DNA, and Provide Additional Tools for Evolutionary Studies.” Microbiology. 151(3): 653-663. DOI: 1099/mic.0.27437-0
- Wiedenheft, B., Sternberg, S. H., and Doudna, J. A. 2012. “RNA-Guided Genetic Silencing Systems in Bacteria and Archaea.” Nature. 482(7385): 331-338. DOI: 1038/nature10886
