CRISPR — The Gene-Splicing Technology — Part 2
CRISPR technology, as I mentioned before, was first discovered in 1985 by the researchers in Osaka University. Nonetheless, it was first used as a gene tool in 2012, by Jennifer Doudna of UC Berkeley. I have given a short timeline of CRISPRs journey from 1985 to 2020 in the image below
Concept:
The concept of Gene editing using CRISPR technique consists of four steps:
- Target the gene which needs to be edited
- Slice the gene at the point of target
- Silence the gene to prevent the self-repair which might cause erroneous results
- Insert the healthy gene at the target point.
Gene editing, and mutation has led to many other similar tools using CRISPR. A few to name are CAMERA( with Cas-9), DETECTR ( Uses Cas -12) [UC1] and SHERLOCK( Cas-13) . I want to discuss CAMERA, because it deals with bit level cellular modification using computing. (Sorry, can’t get rid of computing, highly difficult to stay apart too long).
CAMERA:
CRIPSR Mediated Analogue Multi recording Apparatus is a cell data recorder, which stores the cell’s history. The working concept is simple. The guide RNA is slightly tweaked in such a way to make it active in an event of a viral infection. Once activated, the RNA directs a modified Cas- 9 enzyme( changes to protein) to the infected DNA sequence and instead of cleaving the DNA, the modified Cas- 9 will swap the bases one at a time[UC2] . ( A for G , or G for C or T for C etc.,) Changing the DNA at cellular level using cellular recorders helps to permanently alter the DNA in a known and predictable fashion. When the cell detects a change in a life event such as a cancerous cell spreading toxins in order to kill and conquer other cells, the cell activates the cellular recorder which performs the process of ‘DNA swapping’.
The cellular recording process also helps the scientists to find out whether a life event has occurred or not, and hence helps in detection of many cellular level diseases, not merely cancer. Hence these cellular recorders are highly valuable equipment for bio technicians as they can record multiple kinds of trigger signals simultaneously. Nonetheless these cellular recorders have limited bandwidth and run out quickly when used to record longer events and then CRISPR base editor takes over the process of cellular recorder and swapping.
The triggering of the tweaked gRNA can be made to happen in a plenty of events such as a specific drug usage, a fall in certain nutrient level, presence of cancer cells etc., The strength of the system is so high it can record multiple events at the same time with extremely low noise to signal ratio. Also, the cellular recorders trigger and a swapping is made to happen only in those parts where the cellular sequence has already been mutated. This is confirmed from the previous recording of the cellular event. This is made possible using another CRISPR tool called DOMINO. As the name suggests, the editing happens one after the other similar to domino pieces falling when one piece is pulled. In fact, DOMINO can be used to make logical operations such as AND, OR and NOT, based on the previous recorded events and the trigger and the target can be made more precise. Also, by counting the number of edits made in the past it is possible to determine the duration and the strength of the trigger too, as the strength of the trigger increases with the total number of edits.
The above 30 seconds video of Cas9 molecule performing the cleavage is mind boggling.
But if you want to learn more, check this 2.22 minutes from MIT. But you might need a subscription to watch this.
There are other forms of gene editing technologies such as transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs). But CRISPR — Cas 9 is by far the fastest, reliable, and precise technology currently known for gene splicing.
Big Benefit:
As I mentioned earlier, the best part of it being, CRISPR — Cas 9 system not merely used to replace the infected DNA, but keeps a snippet of the infected DNA stored between the palindromic CRISPR sequences to retain a genetic memory, in order to help the mutated DNA to identify the same virus, if it attacks once again and prevent the infection. Imagine a cancer patient when he /she will never have a reoccurrence event once cured. This can happen in future, thanks to CRISPR cellular recording of the cell’s history. This adds one more additional benefit to this technology- they can treat any viral infectious diseases, such as a liver condition, kidney problems or any other infected organs and the diseases need not necessarily be caused by a genetic disorder. CRIPSR method is better than using radiation or mutation using chemicals, because of its precision and mutating efficiency with which the infected DNA is identified and replaced as against using radiation technique, where, identifying [UC3] the infected DNA is the most difficult part and the gene mutation quantification is nearly impossible, as the scientists were unable to clearly record, how many gene were destroyed and how many were replaced.
Applications of CRISPR technology:
· Medicinal Cure: A range of medical conditions can be treated using this novel technology — the list includes blindness, genetic blood disorders, cancer, Huntington disease, Hepatitis B, high cholesterol and even diabetes. Though currently the patent has been given to edit the non -reproductive cells, it is possible to edit reproductive (germline) cells too using this technology and hence once the ethics committee [UC4] clears it off, then it is possible to make designer babies too, using this super tech. CRISPR cas9 is used in many other applications, other than to cure human diseases.
· Food Industry: It can be used to prevent the good bacteria, contained in foods like yoghurts, cheeses, sour dough etc., from getting infected and thus increase the shelf life of these products in the future. Also this gene slicing and mutation can be used to cure food allergies, by targeting those parts of the plant gene eg. wheat gene, which are responsible for gluten allergies. Though there are many legal restrictions to conduct research on genetic mutations of plants as rewriting the genetic sequence might change certain properties of the known food, which may become harmful through consumption for long duration.
· Pets: The technology is curing diseases in pets such as pure breed dogs and cats. Even the golden fishes might get a makeover to show different colors, if the FDA approves their genetic mutation.
· Agriculture: An UK based company named Tropic Biosciences is experimenting in mutating a coffee bean gene sequence. The idea is to remove the caffeine from coffee without losing the taste of coffee. If this succeeds, it is a blessing in disguise for many coffee lovers who hate the taste of decaff, but cannot take 8 cups of routine coffee. Similarly, there are efforts to mutate vegetables like tomato to make them naturally spicier. They can be used as a part of fertilizer to control pests using gene drives. Gene drives are methods through which a species can be rid of a specific gene for generations to come. CRIPSR can make this happen and stop spreading infectious diseases which are generally spread through insects, pests like mosquito, flies etc., and save our crops and our health. Though gene drives are ethically debated still, if it can be proven to have enough benefits, then they will be adopted universally in agriculture industries.
· Sports: Another application includes in sports arena, such as creating more athletic racehorses by identifying and mutating the horse gene which is responsible for speed and elegance, thus altering the genetic sequence of Race Horses for ever.
· Farm Industry: Healthier farm animals using gene mutation, better biofuels using gene engineering, better food crops and faster harvests by introducing more traits responsible for increased oil content through faster carbon- di — oxide absorption by the plants during the process of carbon conversion into plant biomass can all be made possible through genetic engineering.
· Pharmaceutical: CRISPR can be used in pharmaceutical industries too for efficient creation of drugs, through modifying the molecular structure of certain organic compounds, used in essential drug manufacturing and also the cost and availability of certain essential drugs can be obtained by increasing the yield of certain drug precursors/ Eg of a drug precursor is phenyl acetic acid which is used as a precursor in the production of Penicillin.
Future of this technology:
It might take at least ten years (Possibly in 2031) from now on for this technology, to be used regularly to cure human conditions. Though precision is one of the biggest strengths of this CRISPR — Cas9, it is not 100% precise, there are still small percentage chances where the target might not be the intended infected DNA sequence. Hence many efforts and research are being used to make this 100% precise. The problem of ‘Off target‘, binding is due to the idea of the gRNA finding the same sequence of DNA, which is the complementary base pairs of the 20 bases of the gRNA, elsewhere in the body and cas-9 might erroneously perform the slicing at the wrong sequence. Also with the current technology, the cas9 protein is supposed to edit two strands of DNA, nonetheless researchers are trying to make it cut only one strand, enabling the idea of two Cas9 enzyme and two gRNA to be in the same place to perform a gene slicing, which makes it more precise.
Conclusion:
As I mentioned before, the discovery of CRISPR- Cas 9 has led the researchers to make multiple tools for genetic modification. Genetic engineering has seen a new realm with this discovery and the realm has endless possibilities and enormous benefits. Once the ethical and legal implications are worked out amicably, this technology will start reaping more paybacks, as this research, cost a fortune.
[UC1]Cas- 12 and Cas-13, the inferior cousins of Cas-9 are quite strong even after they reach their target DNA and hence may be extremely difficult to use them for precise genetic mutation or editing.
[UC2]Say for example, AGC gene gives one instruction, while CGA gene might give a different one. If these four letters (A,C,G,U) can be stored as a hexa-decimal value (two numbers can be stored as two bytes of 16 bits), then it is possible to edit this value at bit level by either mixing rows, shifting columns or any other transformation possible. Then the A can be changed to C or G or U. So in cases where A goes rogue, then the gene can be edited and altered with another alphabet by using computing systems. In other words, by altering the nitrogenous pairs in the RNA sequence or a DNA sequence, it is possible to remove the infected part of the cell and replace it a completely new sequence obtained using software coding. Thus, introducing genetic mutation with a nitrogenous pair from a different cell. And this happens at bit level, hence it is more precise than CRISPR and it is considered an improved form of the same. Though the bit level is still under research stages, there is much potential to it in the future.
[UC3]The clear analogy can be blasting a whole wall using TNT , instead of targeting a single brick using a hammer.
[UC4]Because the genetic mutation caused by this technology will be passed from generation to generation, the ethical implications are more in this case.
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References for the 2 part series:
· Development and Application of CRISPR- Cas 9 for Genome Engineering by Patrick D. Hsu,1,2,3, Eric S. Lander,1 , Feng Zhang1,2, of
1. Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02141, USA
2. McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3. Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
· A CRISPR method for genome engineering by Royce Wilkinson and Blake Wiedenheft, Department of Immunology and Infectious Diseases, Montana State University, Bozeman, MT 59717, USA
· https://singularityhub.com/2019/09/04/cellular-computers-get-a-boost-with-crispr/
· https://www.msdmanuals.com/home/fundamentals/genetics/genes-and-chromosomes
· https://courses.lumenlearning.com/microbiology/chapter/structure-and-function-of-rna/
· https://courses.lumenlearning.com/microbiology/chapter/protein-synthesis-translation/
· http://biology.kenyon.edu/courses/biol114/Chap05/Chapter05.html
· http://www.brooklyn.cuny.edu/bc/ahp/SDPS/SD.PS.polynuc.html
· https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1005891
· https://www.genenames.org/about/guidelines/
· https://www.yourgenome.org/facts/what-is-crispr-cas9
· https://www.sciencedirect.com/science/article/pii/S0168945215300686
