Oct 29, 2021
CRISPR — The Gene-Splicing Technology — Part 1
Abstract
CRISPR is a recent technology which edits the human gene and accordingly replaces the infected rogue cell with an uninfected cell from a different part of the human body. This is done on a bit level by changing the human genome sequence. [UC1] CRISPR is the repeated sequence of DNA of many bacteria. CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR- Cas 9 is a gene editing technology.
As this is a completely new topic for many technologists, even those in ‘MedTech’, I am planning to do it as a two-part series. In this Part 1 — I have introduced the basics about the double stranded human DNA, its single stranded derivative RNA, and the CRISPR- Cas 9 technology and in the next Part -2, I will explore the CRISPR technique in detail, along with its tools and applications.
Before CRISPR was introduced in 2012, as a genome engineering tool in the research paper, by Jennifer Doudna of UC Berkeley, CA and Emmanuel Charpentier of Umea University, Sweden, many scientists were of the opinion about having excessive knowledge about the human genome which were rarely utilized as a means to cure human diseases, especially those which are genome related. But with the invention of CRISPR, a great breakthrough has happened in the field of medical studies and medical cure.
‘A genome is an organism’s complete genetic signature; it is the palate of information an organism can call on in order to ensure its own survival and growth. The genome is the collection of information that an organism can pass on to its offspring before birth.’
De-mystifying Genomics, Medical Research Council, UK
Cells — A detailed Exploration
Humans are made up of trillions of cells. I often think that the most difficult job on earth is the study of humans- cell by cell. The notion is because of the following fact — ‘Study of human cells has been happening for centuries, but still, the best of the medical brains, have not succeeded in creating a complete organism, by putting it together cell by cell’. The cell-by-cell technique, is called cellular biology, which is a discipline of study in molecular biology.
A cell[UC1] is the basic building block of any living organism- human, animal, or plant. The important function of a cell is to build tissues. The tissues form into organs and many organs put together having different functionality end up as a whole human being or animal or a plant. There are other functions of a cell[UC2] such as movement in and out of the cell using active and passive transport of small molecules such as oxygen(in) and carbon-di- oxide(out), reproduction through cell fusion(meiosis), growth through cell division(mitosis), produce the energy for their survival and create metabolic reactions by either breaking down the molecules(catabolism) or adding the molecules together to make bigger molecules (anabolism).
Cell Types — Prokaryokes and Eukaryotes
Cells [UC3] are of two basic types — Human Cells (Prokaryotes) and Animal and Plant Cells (Eukaryotes). They are made of up different parts such as cytoplasm, cytoskeleton, plasma membrane, nucleoid, endoplasmic reticulum, ribosomes, lysosomes and peroxisomes, Golgi apparatus, plasma membrane and finally nucleus. The parts will slightly vary for animal and human cells. In an animal cell, there will be nucleus and in human cells, it’s called nucleoid. In animal and plant cells, the genetic material is contained inside the nucleus, whereas in human cells, it is in the nucleoid where it is free floating. One important difference between the animal, plant and human cell is the cytoplasm. In human cells, it is not in a membrane bound organelle. Apart from few other small differences, the core concept is the same. The various parts are responsible for various functions of a cell.
If you’re interested in learning about more differences, click on the below video link. Within five minutes, they tell you all the basics, you need to learn:
Chromosomes
Besides the small differences, the chromosomes are found inside the nucleus, or the nucleoid and chromosomes hold the genetic material DNA. DNA and RNA are polymers, but the monomer in DNA and RNA are nucleotides which are formed by pentose sugar, when combined with nitrogenous bases and phosphate group forms the polymer DNA and RNA.
The entire genetic content of a cell is called a genome. So human genome is the genetic content carried by each and every cell of the trillion cells, the human body is made up of.
Nucleic Acid — DNA and RNA sequence
Human genome is contained inside a chromosome and is a combination of 3 billion letters. The instructions of the genome on how well it has to operate is written as a DNA sequence. DNA is further split into mRNA and mRNA is split into rRNA and tRNA. The core concept of conversion from DNA to RNA to Protein is referred as Central Dogma.
The above video link gives a bit more clarity into what makes our DNA, if you need more info.
DNA Sequence (genome sequence) consists of only 4 main alphabets — A,C,G, and T. The letter stands for adenine, cytosine, guanine and thymine and they are found in nucleotides.[UC4]
Each of the three-letter combo represents a gene, which is eventually a protein cell. RNA sequence also has four letters but has ‘T’ replaced by a ‘U’ for Urasil.
The RNA is extremely similar to DNA, as it is obtained from DNA, though they are few differences . The various instructions are formed by using different sequences of the same four letters and they are used to make proteins.
Amino Acids
The instructions generally consist of three letters out of four nucleotides. They are the nitrogenous base sequence. As I mentioned earlier, the four-base sequence, ACGT of DNA becomes the ACGU in Messenger RNA (mRNA), when it is transcribed into a complex 20 letter language forming the commonly occurring 20 amino acids.
In other words, the 4 nucleotides monomer in human cell is translated into 20 different amino acids.
The complex form is derived by the various combinations of the 3 letters.
A set of three nucleotides are formed to make a codon) thus giving rise to 64 possible words, more than sufficient enough to represent the 20 amino acids[UC5] .
CRISPR — The process of Gene editing
CRIPR manipulates, transforms or edits any of part of genome in any living organism such as fruit flies, mice, plants etc., not merely human genome, though this simple, easy and cost-effective process is extensively used in human medical studies. This is done by destroying the diseased virus genome and replacing the infected sequence with a healthier sequence, thus preventing the virus genome from replicating itself and affecting more cells in the body. In this way, it is possible to provide an adaptive immunity defense to the human body’s bacterial and archaeal cells[UC6] . They function by accumulating the array of spacers or fragments of DNA from previous viral attacks in the human prokaryotic host [UC7] cell.
The success of this technology is due to its precision. The CRISPR technique helps the scientists to target a specific gene in the human gene sequence and leave the rest to be as they are. This is usually done for those viruses which are already encountered by the human body.
CRISPR- Cas 9 — Technique
This is done using two molecules:
1) The Cas -9 Protein which cleaves the foreign infected DNA: For the Cas-9 enzyme to do the gene slicing at the point of viral attack, a CRISPR RNA also referred as ‘gRNA’ is used to identify the infected gene. The gRNA is designed to target and bind to a specific DNA sequence by using RNA sequence of 20 base codons which are complementary [UC8] to the target DNA sequence and hence making it easier for the gRNA to find the target sequence. The complementary sequence are anticodons. Anticodons are nitrogen base pairs( U and C) belonging to tRNA, which are made to pair the single stranded mRNA (or a double stranded DNA which is a codon pair.
2) CRISPR array which performs the job of a virus recognition key and repairs the cut strands of DNA by introducing mutation: This CRISPR array consists of series of spacers, called ‘Protospacers’ and short pieces of DNA, which originates from the Viral DNA and which matches the corresponding parts of the infected DNA sequence. Cas9 enzyme is equipped with PAM (Proto-spacer Adjacent Motif) which is responsible for the efficient searching of the target DNA. PAM is also responsible for triggering the transition from target binding to DNA slicing. The number of spacers is estimated in order to maximize its protection against a viral attack. When the virus attacks the bacterial or the archaeal cell, the spacer from the virus is integrated into CRISPR array and this adapted array is transcribed to make the RNA, which consists of 20 bases of complementary sequence, which acts as the gRNA.
UC1]The cells are of different types and different shapes. The male sperm cell of a human is shaped as a tadpole, a female reproductive egg cell is oval in shape, the nerve cells are in the shape of a tube, muscle cells are tubular in shape, the blood cells are round in shape, the eye cells are in the shape of rods and cones, bone cells come in different shape depending on their functionality. Irrespective of its shape, all cells have a somewhat similar structure and almost the same functions.
[UC2]They also contain hereditary material which humans carry from their ancestors. For more info :
https://ghr.nlm.nih.gov/primer/basics/cell
[UC3]This all may sound like high school biology, but the main reason, I am including it as a part of this article, is to emphasize the importance of human genome and how it is beautifully placed inside the cell.
[UC4]This is a basic block of a DNA or RNA. It consists of pentose sugar, Nitrogenous Bases and a Phosphate group. There are three nitrogenous bases — Pyrimidine (Thymine, Cytosine, Urasil) and Purines ( Adenine, Guanine), Pyridine (Pyridine Nicotinamide)
[UC5]Human DNA coding and encoding is the most difficult task ever performed by the computer scientists. More than 100 million brilliant minds from 5 different countries, UK, US, Germany, China and Japan worked on this decoding project for 13 complete years in order to finish the task. The Human genome project was co-ordinated by US National Institute of Health (NIH) and US department of Energy. As per the project results, humans have about 20,000 to 25,000 genes. The project was started in 1990 and finished in 2003, two years before the completion date. This is one of the greatest medical achievements made in the field of human genome. If you still want to know more about DNA, RNA and Amino acids , click on the following link:
https://www.ncbi.nlm.nih.gov/books/NBK22358/
[UC6]Archaeal cells are similar to bacterial cells , except they are found in extreme environments such as springs, deep oceans etc., Introduction of archaeal cells when the bacterial cells are infected, heals the body , because archaeal cells are non pathogens , while bacterial cells are pathogens, which are mostly responsible for bacterial infectious diseases which can be cured by anti-biotics. For more info on how bacterial and archaeal cell differ from each other, refer to this link below:
https://biodifferences.com/difference-between-archaea-and-bacteria.html
[UC7]This is the human cell and it is divided into two types : Bacterial cell and archaeal cell. The genetic material is not contained within a cell nucleus , unlike a eukaryotic cell where the genetic material is very much found inside the nucleus of the cell. Refer to the video link provided above to check the differences between prokaryotes and eukaryotes.
[UC8] This is also referred as of control of a gene expressions. Gene expressions is due to the difference in various genes based on appearance and functions. Gene expression depends on the type of tissue, the age of the person, the presence of specific chemical signals, and numerous other factors and mechanisms. Knowledge of these other factors and mechanisms that control gene expression is growing rapidly, but many of these factors and mechanisms are still poorly understood.
