Antonio Regalado, a senior editor for biomedicine for MIT Technology Review, wrote an article in 2014 that described CRISPR as the biggest biotech discovery of the century. This was not stretch. 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. There are even studies exploring how it can render the human immunodeficiency virus unable to replicate inside an infected immune cell.
Understanding CRISPR Gene Editing
CRISPR gene editing is a genetic engineering technique based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. It is fundamentally a search-and-replace function for DNA that could be used to pinpoint and disable genes or change their functions by deleting or replacing targeted DNA letters or predefined sequences.
Defining and Understanding Gene Editing
Precision gene editing has moved from lab benches to real-world breakthroughs in health and biotechnology
Genome editing or gene editing with engineered nucleases is a precision genetic engineering technique that uses molecular scissors to insert, delete, or replace specific DNA sequences. In medicine, it effectively reprograms the body by correcting genetic disorders or inducing targeted mutations to develop responses against particular illnesses.
Several gene editing methods have been developed and tested over the last decade. One of these involves using artificial nucleases such as zinc finger nucleases or ZFNs and transcription activator like-effector nucleases or TALENs. These methods are capable of inducing targeted mutation by inserting, deleting, or editing specific and predetermined DNA sequences.
The applications of ZFNs and TALENs have yielded significant outcomes. These range from targeted genetic modifications in plants designed to enhance resilience and boost agricultural yield, to innovative therapeutic approaches in humans aimed at managing or treating a variety of genetic disorders through precise gene editing via DNA alterations.
However, despite their potentials, Koyanagi et al. reiterated that both ZFNs and TALENs remain somewhat difficult and time-consuming to design and develop. Researchers Jennifer Doudna and Emmanuelle Charpentier later discovered in 2013 another gene editing method that involved using the CRISPR system. It was deemed simpler to design and develop.
Other researchers have investigated further the properties and potentials of CRISPR. These studies have fueled optimism for employing this gene editing approach across diverse medical treatments and biotechnological innovations aimed at addressing complex genetic challenges. Some have even shown potentials in treating challenging medical conditions.
History of CRISPR Discovery and Research
What started as puzzling DNA clusters in bacteria became the foundation for one of the most powerful genetic tools in science
It was in 1987 when Yoshizumi Ishino and her colleagues at Osaka University in Japan published a paper detailing the sequence of a gene called iap found in the gut of E. coli bacteria. They sequenced some of the DNA surrounding this gene to better understand its function and discovered that near it were clustered repeats or identical segments of DNA.
The function of these repeats was not clear at this time. Other independent studies also reported the presence of these clustered repeats present in other bacteria and archaea. Their findings revealed that each repeat sequence was separated from the others by DNA spacers. Moreover, unlike the repeat sequences, the DNA spacers had a unique sequence.
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 in their 2002 paper. They further noted a collection of genes accompanied the CRISPR sequences. They called these genes CRISPR-associated genes or Cas genes.
What caught the attention of the researchers is the these Cas genes encoded putative nuclease or helicase protein. These are enzymes that could cut or unwind DNA. Jansen et al. were unable to explain the reason behind the encoding of these enzymes. They were also unable to understand and explain why these Cas genes always at next to the CRISPR sequence.
Three separate studies from separate researchers in 2005 noticed something odd about CRISPR spacers. These spacers resembled viral DNA and extrachromosomal DNA such as plasmids. The three groups drew several conclusions. C. Pourcel et al. concluded that 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. This implied a relation between CRISPR and immunity against targeted DNA. Findings by A. Bolotin et al. also suggested that the spacer elements in CRISPR represent traces of past invasions by extrachromosomal elements.
The study conducted by A. Bolotin et al. further advanced the hypothesis that CRISPR functions as a cellular defense mechanism. This mechanism grants immunity against phage infections and the expression of foreign DNA elements by producing anti-sense RNA molecules that can specifically target and neutralize invading genetic material.
The Function of the CRISPR/Cas System
A microbial adaptive immune defense that archives past encounters and turn them into precision weapons against future invasions
Further studies confirmed previous hypotheses. It turned out that CRISPR works together with Cas and crRNA to form the naturally-occurring defense mechanism CRISPR/Cas system. Researchers J. A. Doudna and her team described CRISPR as an essential component of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea.
It is important to remember that CRISPR consists of distinct DNA segments in bacteria or archaea characterized by short repeated base sequences. These repeats are separated by intergenic spacers or DNA spacers, which are DNA fragments derived from previous encounters with plasmids or viruses, effectively serving as molecular immunological records.
The entire CRISPR/Cas system essentially operates as an intricate molecular complex consisting of CRISPR RNAs and CRISPR-associated proteins. These components work together to recognize and bind to complementary sequences within invading viral or plasmid DNA and then cleave and degrade them to prevent harmful foreign genetic infiltration.
Doudna et al. explained that the CRISPR-mediated immune systems work by specific detection of sequences and silencing of foreign genetic elements. 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.
This foreign DNA insertion produces the CRISPR/Cas system. The system represents all previous exposure to plasmid and viral genetic materials. Moreover, as an adaptive immune system of prokaryotic species, it is used the obtained foreign genetic material to turn Cas enzymes into a targeted immune response mechanism alongside RNA targeting.
Hence, when a microbe once again encounters a plasmid or virus it has previously recorded, the crRNA guides the adaptive immune response and specific recognition process by binding to the matching DNA sequence. The Cas enzyme then cleaves or cuts the foreign DNA into fragments. This effectively blocks replication and halts infection.
The CRISPR/Cas system is a sophisticated prokaryotic adaptive immune mechanism that confers resistance to foreign genetic elements, particularly plasmids and viruses, through RNA-guided genetic silencing. Moreover, to an extent, the CRISPR array functions as a molecular archive, preserving a record of previous encounters with foreign DNA.
Application of CRISPR as a Gene Editing Tool
Jennifer Doudna and Emmanuelle Charpentier turned a microbial immune protein into the sharpest scalpel in genetics
The CRISPR/Cas system has emerged as a promising genome editing tool because of its ability to insert, delete, or replace DNA sequences with precision. MIT Technology Review recognizes American biochemist Jennifer Doudna and French molecular biologist Emmanuelle Charpentier for introducing CRISPR/Cas as a revolutionary technology
Doudna and Charpentier worked together to investigate the molecular workings of a CRISPR/Cas system that employs Cas9. This protein is derived from the Streptococcus bacteria. Their research was intended to establish how Cas9 could be repurposed from a bacterial immune defense into a programmable genetic editing mechanism and gene editing tool
Their experiments revealed that Cas9 could cut DNA sequences at specific locations when paired with laboratory-designed guide RNA molecules. These RNA molecules are engineered to match targeted DNA segments and enabled researchers to direct Cas9 precisely where they wish to induce changes within the genome of a particular organism.
From these findings, Doudna and Charpentier concluded that the CRISPR/Cas9 system holds vast potential as an RNA-programmable editing platform. Because the guide RNA can be tailored for virtually any sequence, researchers envision applications in nearly all organisms, offering possibilities for medicine, agriculture, and fundamental studies.
Both scholars and startups are actively expanding CRISPR research. Studies have shown its ability to activate or silence genes, model diseases by knocking out specific genes, and even remove infections at the cellular level. Emerging applications include treating genetic disorders, combating cancers, and engineering species with predefined traits.
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