Biology's 'Molecular Scissors': Treating Genetic Diseases and the Future Revolution Through CRISPR-Cas9
Born from bacterial immunity, this technology is now capable of curing incurable diseases by modifying human DNA.
In the history of biology, there are certain discoveries that possess the capability to change the future of entire humankind. CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) is one such revolutionary technology, commonly known as 'molecular scissors'. This technology empowers scientists to modify the DNA of living organisms with extreme precision. According to Wikipedia reports, this discovery was awarded the Nobel Prize in Chemistry in the year 2020 to Emmanuelle Charpentier and Jennifer Doudna.
CRISPR is fundamentally a defense system found in nature. It is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. When a virus attacks a bacterium, the bacterium saves a small piece of that virus's DNA into its own CRISPR sequences. This process provides the bacterium with a kind of 'memory' or acquired immunity to fight against the same type of virus in the future. According to Source 1, this natural protective shield is found in approximately 50% of bacteria and 90% of archaea.
- Spacer acquisitionWhen a virus attacks, the bacterium captures a piece of its DNA (spacer) and stores it in its own genome.
- RNA processingFrom the stored DNA, pre-crRNA is formed, which is cleaved into small mature crRNA pieces.
- InterferenceIn the future, when the same virus attacks again, Cas9 and crRNA together cut the virus's DNA.
The history of this system is quite fascinating. In 1987, Ishino and his colleagues first observed these unusual repetitive sequences in E. coli bacteria. However, their function was unknown at that time. In 2001, Spanish microbiologist Francisco Mojica named these sequences 'CRISPR'. In 2005, Bolotin and colleagues discovered that these sequences help in resisting viruses. In 2012, Doudna and Charpentier developed this natural system in the laboratory as a simple tool that can be used to cut any DNA.
To understand how CRISPR-Cas9 works, we have to understand its two main components. The first is Cas9, which is an endonuclease enzyme. It performs the job of cutting DNA. The second part is guide RNA, which works to lead Cas9 to the specific part of the DNA. This process is like a GPS system, where the RNA determines the location and Cas9 goes there and makes a cut.
When the mixture of Cas9 and guide RNA is introduced into a cell, it moves along the long strand of DNA. Cas9 specifically looks for a short signal called PAM (Protospacer Adjacent Motif). For the Cas9 derived from Streptococcus pyogenes bacteria, this signal is 'NGG'. Once the PAM is found, Cas9 unwinds the DNA and if the DNA sequence matches with the guide RNA, it makes a double-strand break, meaning it cuts both strands of the DNA.
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After making a cut in the DNA, the cell's own repair mechanism becomes active. There are two main pathways here: the first is Non-Homologous End Joining (NHEJ), in which the cell directly joins the cut ends. This process often introduces small errors, which inactivates that gene. The second pathway is Homologous Recombination (HDR), in which if scientists send a new piece of DNA along with it, the cell integrates that new piece into the cut region. In this way, we can correct a defective gene or add a new gene.
| Component | Typical size (bp) | Limit (bp) |
|---|---|---|
| Repeats | ૨૮-૩૭ | ૨૩-૫૫ |
| Spacers | ૩૨-૩૮ | ૨૧-૭૨ |
| Guide RNA (Spacer region) | ૨૦ | ૨૦ |
The figures of CRISPR technology demonstrate its precision and capability. CRISPR repeats are generally 28 to 37 base pairs long, while spacers can be 21 to 72 base pairs. The Cas9 protein has a weight of approximately 160 kilodaltons. In 2013, the teams of Feng Zhang and George Church successfully used this technology in human cells for the first time. In 2015, Chinese scientists also conducted experiments on human embryos, which sparked ethical debates.
- DesigningA guide RNA corresponding to the target DNA is prepared.
- CleavageThe Cas9 enzyme cuts both strands of DNA at the specified location.
- RepairThe cell joins the cut part, in which a new gene can be added or a defective part can be removed.
The biggest advantage of this technology lies in the treatment of genetic diseases. According to Source 2, researchers have succeeded in reducing infection by cutting the DNA of viruses such as Hepatitis B and HIV. In addition, clinical trials are also underway for genetic diseases causing sickle cell anemia, cancer, and blindness. In agriculture as well, CRISPR is being used to develop crop varieties that yield higher production and possess disease resistance.
However, there are also certain challenges associated with CRISPR. The biggest challenge is 'off-target effects', where Cas9 mistakenly cuts the wrong part of the DNA. This can cause severe problems like cancer. Scientists are now developing new variants like Cas9 nickase which are safer. Furthermore, the ethical questions of modifying human embryos are also extremely complex, because such modifications can be passed down to coming generations as well.
For a country like India, where the prevalence of genetic diseases is significant, CRISPR is a ray of hope. Since it is an inexpensive and rapid technology, its accessibility to the general public is likely to increase in the future. By 2025, new researches in this field, such as the use of apoNmeCas9, will make this process even more efficient. Ultimately, CRISPR is not merely a scientific tool, but it is a marvelous art of utilizing nature's oldest defense system for human welfare.
