It's time to introduce what is without a doubt the most promising biotechnological approach of the last 10 years. In earlier lessons, we looked at a few biotechnological principles. The CRISPR-Cas9 system. This technology has revolutionized molecular biology by enabling precise and site-specific gene editing, offering an unparalleled level of control in manipulating genetic information. What are the applications for this, and how does it function mechanically? Now let's take a deeper look. Putting some historical background first.
The Discovery of CRISPR
Atsuo Nakata and his group of researchers from Japan's Osaka University in 1987 initially revealed the existence of Clustered Regularly Interspaced Short Palindromic Repeats referred to as CRISPR, within the genome of Escherichia coli. These are short, repetitive DNA nucleotide sequences that are present in prokaryotes' genomes. Similar to how we refer to words like "racecar or kayak" as palindromes because they are the same whether read forwards or backwards, these sequences are also called palindromic repeats because they are the same when read from 5' to 3' on one strand of DNA and from 5' to 3' on the complementary strand. This is also observed in Archaea and Gram positive and Gram-negative bacteria, raising the obvious question of whether CRISPR is applicable to these organisms and motivating research for a while.
Prokaryotes were the first to demonstrate the use and significance of CRISPR later on, in the mid-2000s. It turns out that these prokaryotes' adaptive immunity, which defends themselves against assault by plasmids, bacteriophages, and viral DNA, is largely dependent on the CRISPR system. Yes, despite what might seem impossible, even unicellular bacteria have a very primitive immune system. If you recall the Immunology studies that adaptive immunity is the immunity that an organism develops following exposure to an antigen, which can come from a vaccination or from a virus.
Humans who receive vaccinations, for instance, develop a type of adaptive immunity because the body produces antibodies in reaction to antigen exposure, which aids in the development of immunity. For bacteria, this operates in the following manner. The distinct sequences, known as spacers, that are tucked between palindromic repeats are segments of foreign DNA that come from mobile genetic elements or MGEs, such as bacteriophages, transposons, or plasmids that have previously infected the prokaryote. These segments do not belong to the bacterium. Sequencing the spacers in the CRISPR system confirmed this, supporting the theory that it may be a defense mechanism used by bacteria to identify foreign DNA segments.
In order to create CRISPR arrays, bacteria that are infected with viruses take up tiny bits of foreign viral DNA and incorporate it into the CRISPR locus. These are made up of palindromic repeats, or duplicated sequences, found in the bacterial genome, surrounded by variable sequences, or spacers, originating from foreign genetic components. In this manner, bacteria are able to remember an earlier infection. Consequently, even though CRISPR was first identified as a genomic component of bacteria in Archaea, it has sparked the development of a genome editing technique that can be used on a wide range of eukaryotic organisms. However, before we can do that, we must first comprehend how CRISPR functions in prokaryotes. This is because knowing how it works in its native state will help us comprehend how it is used to modify the genomes of humans and other creatures.
The Mechanics of CRISPR-Cas9
Let's examine a specific Streptococcus bacterium that a bacteriophage is attacking. A portion of the viral DNA can be integrated into the bacterial genome when it is injected into the cell; as we previously explained, this integration will occur in between the repetitive palindromic sequences. We will now refer to this as a spacer. Thus, we have three distinct spacers, possibly originating from three distinct viruses sandwiched between the palindromic sequences that are repeated. We now possess an apparatus known as CRISPR array. Although this longer strand is known as pre-crRNA, transcription of CRISPR array can result in CRISPR RNA, also known as crRNA. Then, the Cas9 protein becomes active. The term "Cas" stands for CRISPR-associated nuclease protein. Nucleases are known to be enzymes that can cut DNA at particular nucleotide links, much like a pair of scissors. We will be focusing on Cas9, one of the nucleases present in Streptococcus pyogenes, which is one of the most well-studied and well-characterized CRISPR-associated nucleases proteins, inside the bacteria.
Now tracrRNA molecules are present in addition to Cas9. These can anneal to the palindromic repeats since their parts are complementary to them. Thus, we end up with a complex made up of pre-crRNA segment, a tracrRNA, and a Cas9 protein for each spacer and palindromic repeat. The strand between these complexes will then be broken down by a different enzyme known as ribonuclease 3 or RNA III, leaving us with individual crRNA complexes that we can refer to as effector complexes. The cell is now prepared to fight against the Intruders whose genome produced that crRNA after these effector complexes have formed. The nuclease enzyme will coordinate if this complex comes into contact with the region of the viral DNA that contains a sequence that is complementary to the crRNA. If it detects a brief sequence that is specific to the viral genome known as a protospacer adjacent motif, or PAM, it will cut both strands of the DNA, a short distance upstream from the PAM. By doing this, it will neutralize the virus and prevent infection because its genome can no longer be correctly transcribed to produce new viral particles.
The Biotechnological Application of CRISPR-Cas9
This provides us with a reasonable grasp of how prokaryotic organisms use CRISPR as a defense mechanism. It's time to comprehend how the foundation of biotechnological application was created from this phenomenon. This all started in 2012 when French microbiologist Emmanuelle Charpentier and molecular biologist Jennifer Doudna of the University of California, Berkeley first suggested that the bacterial CRISPR-Cas9 system could be used as a programmable toolkit for genome editing in humans and other animal species. For their efforts, they were awarded the 2020 Nobel Prize in Chemistry. So how may this approach be used to do genome editing?
First, it's important to realize that tracrRNA and crRNA are two different molecules found in bacteria. The discovery that these molecules' responsibilities could be united into a single molecule by joining them with a linker to create single guide RNA, or sgRNA, which can be produced in the lab, was the first significant advancement. This two-component system can cleave DNA in the same way as the three-component system in the bacteria if the sgRNA forms a complex with a Cas9 protein. This meant that any sequence of about 20 base pairs could then be identified as a target for editing; all that needed to be done was create the corresponding sgNA and insert it into a cell together with the Cas9 protein, which was obtained from Streptococcus pyogenes. When the complex forms, it will create the DNA until it reaches the correct sequence in conjunction with a PAM sequence. Once this happens, binding will take place, cleaving the DNA at the precise side that is needed. Each of the two domains that make up Cas9 will cut a single DNA strand. The target DNA undergoes spontaneous repair after the incision thanks to this mechanism.
There are two ways to repair cleaved double-stranded DNA through non-homologous end joining, also known as NHEJ or homologous-directed repair, also known as HDR. The NHEJ process ligates DNA directly to repair double-strand breaks without the use of a homologous template, which is a DNA strand that is comparable in sequence and can serve as a template. Additionally, some sequences may be inserted or deleted needed at the joining ends via the NHEJ mechanism, resulting in the creation of what are known as indels. Indels are DNA strands that have nucleotide sequences added or removed. As a result, NHEJ generates DNA strands with irregular sizes. The NHEJ mechanism we just covered is more common in the eukaryotic domain, whereas the second route of repair, the HDR pathway, is frequently seen in bacterial and archaeal cells. Despite being more intricate than NHEJ, the HDR procedure makes use of a homologous DNA template. In order to incorporate additional DNA fragments, the homologous DNA template must have similarity to the nearby sequences around the site of cleavage. The repair procedure is guided by the template, which also reduces the likelihood of mistakes. Unlike NHEJ, the HDR process maintains homogeneity in the size of the resultant double-strand DNA since there is no nucleotide sequence insertion or deletion.
The Applications of CRISPR-Cas9
That concludes our discussion of the CRISPR genome editing technology's workings. We now turn our attention to the possible uses, which have only grown since Doudna and Charpentier proposed that CRISPR may be used to change the genomes of humans and other animals. CRISPR has a wide range of possible applications and involves using it as a genetic screen to distinguish between genes in various cells.
The most well-known use is in immunotherapy for cancer. This procedure involves the genetic modification of immunological T cells, a subset of white blood cells that combat illness, using CRISPR technology. In particular, these T cells are taken out of the patient and altered so that they are better suited to identify cancer cells and eliminate them when the patient's body is reintroduced.
The human immunodeficiency virus or HIV is the cause of Acquired immunodeficiency syndrome, or AIDS, which has been treated with CRISPR. Viral replication can be inhibited by standard antiretroviral treatments. However, once the virus transforms into its proviral form, standard treatments are unable to eradicate it. The provirus is housed within immune cells and uses the machinery of immune cells to replicate itself. Because the immune cells do not specifically target the proviral latent reservoir, there is a danger of viral rebound or the return of the disease. CRISPR has shown great promise in the development of an assay to identify SARS-Cov-2 infection, the source of the present worldwide pandemic, in addition to cancer and AIDS.
CRISPR holds the promise of curing a variety of diseases and preventing the inheritance of gene-linked diseases, despite the vast range of ethical problems and potential hazards associated with genetic editing of somatic cells and human embryos and their implantation into human wombs. Furthermore, the use of CRISPR technology for genome editing in plants opens up the option of incorporating particular qualities, increasing crop productivity, improving phenotypic, or observable attributes, and making plants resistant to specific illnesses.
It will be interesting to watch which of these important illnesses and problems get resolved first, indicating the start of a new chapter in the history of molecular biology, because there are so many fascinating possibilities for this innovative new technology.
