In 1987, while just casually fiddling with yogurt bacteria, a group of researchers stumbled upon a pattern in its DNA. There seemed to be a particular sequence of base pairs, or DNA letters, that repeated periodically in a portion of the yogurt bacteria DNA. Perplexingly, the repeated portion was like a marker or spacer, and between two such series, the bacteria had stored the DNA of viruses that it had encountered in the past. Thus, like a memory, the bacteria knows immediately when it is attacked by such a virus. This helps the bacteria to quickly initiate an immune response. The researchers named the markers in the DNA as Clustered Regularly Interspaced Short Palindromic Repeats or simply “CRISPR”.
By 2002, CRISPR started appearing in all sorts of bacteria. It was found that the bacteria can transmit the CRISPR DNA to the next generation pretty efficiently.
It was clear by 2008 that the CRISPR DNA is just a storage system but the cool part is the CRISPR Associated Protein or the Cas protein, which has the capability to find foreign viral RNA or DNA and cut it like a pair of scissors all by itself.
The CRISPR-Cas protein was studied by this year’s Nobel Prize winners: Jennifer Anne Doudna, an American biochemist and Emmanuelle Marie Charpentier, a French professor and researcher in microbiology, genetics and biochemistry.
The CRISPR-Cas system is a molecular tool that lets scientists edit DNA. It has two parts: an enzyme that cuts the DNA at a specific point, and an RNA guide that helps that enzyme to find and reach the right place to cut.
The breakthrough was when the research duo realised that the Cas protein can be extracted from the bacteria and used as a Gene Editing tool. They published their research paper in 2012 and patented the editing system. In a nutshell, the CRISPR Editing system/process works through the CRISPR-Cas protein, and a popular choice is the Cas-9 Protein. A predetermined short RNA sequence (called the guide RNA) is fed into the Cas protein, which then searches the full genome of the cell along with DNA and RNA in the cytoplasm (body) of that cell. Once a matching sequence is found, the Cas protein binds to that gene, and precisely cuts it, just like a pair of scissors. In real life, all genes get damaged from time to time, so there are enzymes and various mechanisms which constantly fix and repair the genes. Naturally, the mechanisms of the cell try to recover the part of the gene cut by the Cas protein. This is when a new sequence is externally introduced into the cell, and it gets attached to the gene automatically.
Advantage of using CRISPR
CRISPR can thus be used to edit genes at very quickly and efficiently. Previous generations of gene editing tools were remote, in the sense that it took very long time even for simple edits, and to be able to use those methods, one had to send the genes to an external company, that took days to edit all the genes in all the cells. With the discovery and adaptation of CRISPR, researchers can edit genes within minutes, that too inside their labs. The CRISPR system can be used inside living cells without hindering its normal functioning. The CRISPR mechanism can be inserted into a virus, and using that virus, the genome of a whole organism can be changed.
CRISPR has accelerated microbiology, especially gene-based research to say the least. There were a lot of very hardworking and exceptionally smart researchers, whose combined effort made CRISPR possible, but Jennifer Doudna and Emmanuelle Charpentier were the crucial masterminds in the research process.
It usually takes around thirty years for a published paper to be recognised and applied to an extent that the authors are entitled to a Nobel Prize. Though, the fact that it took less than 7 years for the CRISPR technology to be realised to its full extent and be used world-wide for cutting edge research is a breakthrough for humanity. Also this is the first time a pair of female scientists have won the Nobel prize in the field of sciences, securing a promising future.
Okay, quick summary:
Yogurt scientists found a pattern in the genome of bacteria that led to speculation and investigation. The CRISPR Cas system was discovered and soon extracted to perform customised cuts in the DNA, and viola! The CRISPR-Cas gene editing system was born. It is a word processor and editor for genes.
With enough research scientists can make horses with horns or maybe even Blast-Ended Skrewts, though, that’s not on the priority list…
So what are the current areas of research where CRISPR plays a trivial role?
In its initial days and even today, CRISPR gene editor is being used to study the effects and purpose of each and every gene in all organisms. It allows precise removal of a specific gene from the genome of an organism in its embryonic form. Now, scientists can observe the changes in the structure and behaviour of that organism as it grows, in the absence of that gene. Thus, by studying the absence of a gene, its function can be inferred and speculated.
Drought resistant crops, better yield from modified seeds, non-browning apples and larger juicier fruits are now a reality thanks to CRISPR.
Some human diseases are caused by a small change in the entire genome, like a single letter change, addition or removal of a few bases. CRISPR technology can be incorporated into regular medicines that target reachable cells, and change the genome such that the disease can be cured. Blood cells are the easiest to reach and application of CRISPR editing in blood cells is quite practical. A major area of research today is to find a cure for blood diseases, cystic fibrosis, HIV, sickle cell anemia, Cancer and blindness through CRISPR medicine.
Genetic cardiovascular diseases, like hypertrophic cardiomyopathy (enlargement of a part of the heart), are caused by a small change in a specific gene. By in-vitro experimentation of CRISPR-modified live tissues, scientists are starting to make out what change to the gene results in that disease. Thus after many trials, customised CRISPR based heart treatment might be available.
Limb regeneration in tadpoles and fat metabolism in flies has been studied with the help of CRISPR. A slightly modified version of this gene editor, in which the Cas-9 enzyme is deactivated, just finds the location of a particular sequence of DNA, without cutting it. This d-Cas-9 enzyme is a marker, which helps to navigate any cell.
Eradicating malaria, yellow fever and dengue can be achieved by genetically modified mosquitoes. Recently, Florida announced to release 750 Million GMO mosquitoes in 2021, with the hope to wipe out Aedes aegypti.
There is even a CRISPR based COVID test!
The seemingly perfect CRISPR has its limitations. For one, it is capable of making a small change at a time in the entire genome of the full DNA sample. Though, several CRISPR systems can be used in parallel in a single sample of DNA.
CRISPR is very precise in cutting and editing genes, but “off-target effects” show up quite often. This is when in the DNA sample, there is a very similar sequence to the one to be cut, and the CRISPR-Cas enzyme cannot differentiate between the two, so both are cut. This leads to unwanted changes in the sample DNA. Off-target effects are relatively rare: on an average 2-3% of the sample is contaminated by these wrong edits. However, in a large sample size, they can easily accumulate and distort the desired result.
All CRISPR genetically modified organisms, from fruit plants to rats, do not transfer the modified genome to their next generation effectively. There are two pairs of homologous genes in almost all organisms (one from mother and father each). And the CRISPR modified gene rarely survives in the wild. Its effects wore out after 2-3 generations, as there is only one copy of the modified gene, but several natural genes in nature. This, however, can be overcomed by the use of gene drives.
CRISPR-modified genes might not survive for even a few generations in the wild, but what if the genes of the next generation could be automatically modified without external intervention? Enter Gene Drives. Through a long and tedious process, the genome of an organism can be modified to produce Cas proteins, which will modify the genome automatically in the next generations. Gene drives are segments of DNA, artificially attached to the genome of an organism, which will produce CRISPR Cas proteins in the coming generations. For example, a single GMO mosquito with a red body and a corresponding gene drive, can transmit the gene drive to all its offspring. Thus when released in the wild, all wild mosquitos’ will be automatically modified to have red bodies.
This has huge implications. Now, humans have the power to change entire species at will, even produce GMO human embryos and rekindle extinct species. There are a lot of moral dilemmas that threaten existence and nature itself.
“Personally, I don’t think it is acceptable to manipulate the human germline for the purpose of changing some genetic traits that will be transmitted over generations.” ―Emmanuelle Charpentier, co-inventor of CRISPR-Cas9
Soon after the power of CRISPR was realised, both Doudna and Charpentier globally called for all human embryo and germline related CRISPR research to a pause. There were several meetings and conferences with international scientists, researchers and stakeholders, which aimed at coming up with all the ethical issues surrounding CRISPR and also to come up with guidelines to prevent the misuse of this powerful technology.
The realm of genetics is quite new, and a lot of research is required to understand, standardise and apply the inner workings and repercussions of CRISPR.
The world of molecular biology and genetics is evolving at such an exploding rate that there are breakthroughs and new inventions every month, all around the globe. CRISPR is a key part in this process. Pressing issues, like finding a viable and effective cure for cancer, are closer to reality than to sci-fi.
“The power to control our species’ genetic future is awesome and terrifying. Deciding how to handle it may be the biggest challenge we have ever faced.”
― Jennifer A. Doudna, A Crack in Creation: Gene Editing and the Unthinkable Power to Control Evolution
This article was written for
Stematix Magazine (http://www.stematix.org/) by Aryan Tiwari