Synthetic Biology-What can we expect...

                        Synthetic Biology-What can we expect:

Synthetic Biology deals with the study of existing biological systems and structures, redesigning them and formation of new biological systems. This field combines knowledge and apply it for the betterment of mankind from a number of fields including; genetic engineering, molecular biology, biotechnology, biophysics, molecular engineering, computer and electrical engineering.

The field of synthetic biology is continuously revolutionizing the world as it provides human with all the necessary knowledge needed for dealing with various problems, diseases and helping mankind to think beyond boundaries and enabling them to devise such techniques and technologies which were considered impossible before.

CONTRIBUTIONS:

Following are some recent contributions by the field of synthetic biology.

1.Chimeric Organisms:

                                        The word chimera is driven from Greek mythology which means a monster having head of a lion, body of a goat, and tail of a snake, ultimately a combination of various living beings. In biology such thing is termed as chimera which contains materials from multiple living sources, it could be a chimeric DNA or a chimeric organism. Now a day scientific community across the globe is working on developing chimeric organisms having human genetic material and capable of producing various human organs which could ultimately be used for clinical trials, testing of various new medicines, and for transplant purposes. At the moment the demand for organ transplant is 4 times the supply. So, successful formation of chimeric organisms having human cells and organs will be helpful to meet this requirement. Multiple experiments were carried before human trials:

   · Inserting rat stem cells in to mice forming rat-mice hybrid.

   ·  Inserting rat stem cells in to pig no positive result.

   ·  Inserting human stem cells in to pig embryo. Positive result and formation of human –pig hybrid.

Procedure for the formation of Human-Pig hybrid

2.Bio-fuels:

                  Petro-chemicals are now becoming an out dated source to power our vehicles.  Continuous CO₂ emissions and global warming issues are forcing man kind to shift towards a viable source. Bio-fuels are the best possible alternative till now to power our vehicles. The formation of bio-fuels is a complex process and greatly dependent on synthetic biology. Formation of such bio-catalysts which can effectively and efficiently convert sugars in to desired products is because of molecular engineering and synthetic biology. Researchers are now struggling to develop more efficient bio-catalysts which can produce high quantity of bio-fuels out of sugars and also do it more cheaply in order to bring the cost of bio-fuels way lower than the traditional petrochemicals. Many developed and highly developing nations are already producing bio-fuels in large quantities and are using it. USA, Brazil and Germany are the top 3 producers followed by China, Argentina, France, Indonesia, Canada, Thailand, and Colombia.

Top 10 Bio-fuel Producers (Courtesy World Economic Forum)

3.Bio-sensors:

                          Bio-sensors are such organisms which are modified to give out certain signals upon completion of some physiological and biological process. These kinds of sensors can be used to detect and quantify certain reactions and biological process. These bio-sensors provide an effective way to monitor cells under in-vivo conditions helping scientists to better understand the survival, migration, and possible tumourigenicity in animal cells.

4.Data Storage:

                           Storing data is a key part of learning and evolving as it enables us to look back in our past and see where we did right or wrong and what is necessary to be done to deal with a specific problem. But to store data for a long time is a challenge. Normal hard drives have a life span of 5-10 years and are unable to store data for centuries, which forces us to make its copies regularly and if one doesn’t do it he’ll lose the data forever. Scientist are working to deal with the problem by creating such thing which can hold data for centuries without any issue. Science is now looking at nature for a solution. Researchers at The ETH Zurich, Switzerland believe that answer lies in natural data storage system of living cells: THE DNA, So compact and complex that a 1gm can theoretically store all data of TECH Giants like Google, Amazon, Facebook. In storage terms 1 gm DNA can store 455 exabytes of information and 1 exabyte is equal to a billion gigabytes.

DNA is a unique way of nature to store data for centuries in form of fossils. Scientist has succeeded in extracting genome of 110,000-year-polar bear and recently a 700,000-year old horse. Robert Grass of Dept. of Chemistry and applied Bio-science said:

       "We have found ways of making DNA very stable, So we want to combine high storage density of DNA with stability of DNA found in fossils"

Future USB's might be made up of DNA

5.Nano-particles:

                          Use of nanotech in various fields of science including biology is increasing day by day. Nanotech in biology has shown some promising results so far indicating the impact it could make on our lives. Many kinds of nano-robots are used as drug delivery systems which deliver a certain medicine directly in to the infected cells preventing the uptake of the drug by healthy cells and avoiding some malfunctioning and side effects of the drug. These nano-robots are enabling researchers to deliver the drugs in a very controlled manner and dosage. Various nano-particles are also being used to reduce anti-biotic resistance. Various nano-particles are under clinical trial for treatment of various diseases. E.g.:

   · Abraxane are the nanoparticle albumin bound paclitaxel used for the treatment of various type of cancers including breast cancer and pancreatic cancer.

   · C-dots are small silica based nano particles which give fluorescence with an organic  dye and is being used for easy detection of tumour cells by surgeons.

   · Nano particles are also used for treatment of eye diseases including cataract and dry eye disease.

Nano-robots directly delivering medicine to individual cells

6.Genome editing:

                               Every single development inside a living organism is managed by its genome all the necessary data is encoded inside the genome. A minor error in this genome can result in to severe and incurable diseases but thanks to genome editing these issues are now less complicated. Genome editing is the thing these days. Everyone across the world is talking about it and the impact it can cause on the human life. Imagine of an incurable disease which can be treated with replacing the error gene with healthy one simply by cutting the first are replacing the latter with it. According to an estimate almost 85-90% of genetic disorders are incurable but with this gene editing we can deal with those issues. Along with treatment of diseases genome editing can also be used for solving many other problems. It can be used in production of more advanced and efficient plants, animals and even humans. It can be used in plants to make them resistive against various diseases without any usage of foreign DNA, improving yield outputs of the plant, making them more adoptive to harsh environmental conditions, etc. Genome editing is an efficient way to manipulate a living being according to our desire and can prove extremely beneficial in near future. Currently the most effevtive genome editing tool is the CRISPR/CAS-9 , others include CRISPR/CPF-1, Zinc finger nuclease, and TALENS.

Classification of CRISPR-CAS System

Classification of CRISPR-CAS System

Ever since its discovery the CRISPR CAS System has become a vital tool in the field of life sciences. What was originally discovered as bacterial immunity system in the bacteria is now being used in the field of life sciences as a broad range genome editing tool. After scientist successfully hijacked this immunity system and turned it in to a genome editing tool, it has opened a new horizon in the field of biological sciences but it was a tough journey. One of the basic challenges faced by the scientists was to characterize various types of CRISPR Systems, their characterization was a big challenge thanks to extensive exchange of CAS genes and gene modules among various bacteria.

Scientists across the globe performed number of genomic analysis in order to establish a classification system capable of broadly identifying various types of CRISPR CAS systems. After that position specific scoring matrices (PSSM) of all the known CAS systems were developed. Then only those CAS locus were considered for further classification which were declared complete. A CRISPR-CAS system was declared as complete if it posses the adaptation modules and complete set of genes required for the formation of interference module. Keeping in view all these findings CRISPR-CAS system was divided on the basis of what type of interference module is encoded by its genes. Those encoding for a multi subunit CrRNA complex were placed in “Class-1” CRISPR systems and those encoding for a single multipurpose interfering module were placed in “Class-2” CRISPR-CAS systems. Each class has further types and subtypes.

The Class-1 has:  

•  Type I
•  Type III
•  Type IV

The Class-2 has:  

• Type II
• Type V

Class 1:

The class 1 systems are described as those types of systems which require a large complex of multiple proteins to conduct the interference procedure. The class 1 has further 3 types The Type I, Type III and Type IV systems.

   v  Type I:

          The type I system is further divided in to a total of 7 subtypes namely sub-type, I-A, I-B, I-C, I-D, I-E, I-F, and I-U. All kinds of type I locus consists of a gene that encodes for the signature protein CAS 3 or its variant the CAS 3’. This furthers promotes the formation of helicase  which causes unwinding of double stranded DNA or the DNA-RNA complex. The helicase is fused with endonucleases which causes the cleavage of target DNA. Many of the subtypes are typically encoded by a single operon which encodes for cas1, cas2, cas3 and genes for cascade complex subunits.

  v  Type III:

             All the type III CAS systems are characterized by the presence of a specific protein called the CAS 10 protein which is the signature for the Type III systems. This CAS 10 induces formation of a multi-domain protein containing palm domain which is the largest subunit of the crRNA-effector complex. The type III systems are also reported to produce a CAS7 and a CAS5 protein subunit. This complex is further fused with specific nucleases which cuts and denatures the enzyme.

  v  Type IV:

              The type IV CAS system is one of the two new CRISPR CAS systems discovered in the recent studies. In this type the “csf1” protein serves as the signature protein. The specific function of this system type is still uncharacterized but how it functions? That has been sorted out recently; it has a multi-subunit crRNA-effector consisting of Csf1, Cas5 and Cas7 protein subunit. In addition to that it also contains a Ding family helicase or a alpha-helicase.

Class 2:

These type of systems are describes as those type of CRISPR-CAS system in which the entire procedure of interference is carried out by a single large proteinic molecule.  The class 2 has 2 types.

  v  Type II:

            Type II system is the most widely studied system because of the famous CAS9 belonging to this type. The signature gene for this system is the CAS9 which encodes for a single multi-domain protein which carries out the interference function. The system also contains CAS1 and CAS2 genes which carries out the adoption stage and is often assisted by the CAS9 protein. The type II system is further subdivided in to 3 subtypes; subtype II-A, II-B, and II-C. The II-A subtype posses an additional “csn2” gene, it has been reported to assist in spacer acquisition. The subtype II-B lacks csn2 but it has a “cas4” gene. The subtype II-C has only three genes the CAS1, CAS2 nad CAS9.

3D model of CAS9

  v  Type V:

            Type V is one of the two newly discovered CRISPR systems (type IV being the other newly discovered system). This system is characterized by having a special gene called the “cpf1” gene, which encodes for a large protein of about 1300 amino acids. This system type was first discovered in prevotella and francesella bacterial species. In similarity to CRISPR CAS9 all the interference process is conducted by a single large protein module named the CPF1. However the cpf1 lacks a n HNH nucleases domain which is common in CAS9 protein. Also the cpf1 is encoded outside the CRISPR-CAS context in several genomes which indicates it to be a possible addition because of transposable elements. In addition to cpf1 it also encodes for a cas1, cas2 and in rare cases cas4 proteins. Unlike other Class II systems which are specific to bacteria the cpf1 has been reported in one archae as well.

 

3D model of CPF1

Conclusion:

                         Till now scientists have successfully classified various CRISPR-CAS system in to 2 classes, 5 types and 16 subtypes, on the basis of their genetic characters and protein modules produced by the genes. This classification is vital for scientists as it will enable them to further enhance their knowledge, easily identifying and placing new CRISPR systems and implementing newly discovered systems for further high tech research.

Mechanism of CRISPR-CAS System

Mechanism of CRISPR-CAS System

Like every living being bacteria and other micro-organisms also face continuous challenges in form of varying environmental factors and pathogens. The biggest enemy of bacteria is the bacteria eating viruses called the bactreriophages. And like every organism bacterial cell has also developed a way to deal with it. With the passage of time bacteria have developed a natural system, a kind of immunity to deal with the invading viruses, and this special system of bacteria is known as “CRISPR”.

CRISPR is a natural immune system embedded inside the genome of various micro-organisms till now about 40% of bacteria and almost 90% of the total archaea, whose genome has been sequenced, has been reported to have the CRISPR-CAS immunity which enables them to fight back the invading viruses and prevent the viral infection. In the beginning very little was known about this immunity system but with continuous advancement in the field of genomics and molecular biology much has been discovered about this system, what it is composed of and how this system works.

Components of CRISPR-CAS System:

The CRISPR-CAS system exists as a part of the bacterial genome which upon the entrance or attack of a virus gets trigged and start producing corresponding products which enables the bacteria to stop viral infection. The CRISPR region of the bacteria is composed of three parts:

1.  CAS genes
2.  Spacer DNA
3.  Palindrome repeats

Image showing 3 parts of CRISPR Locus

Each individual part of the CRISPR region plays its own unique role in order to achieve the overall activity of the CRISPR system.

CAS Genes— These genes encode for special protein called the CAS protein which plays a dual role in the CRISPR system it acts like a helicase as well as endonucleases and is responsible for the unwinding, cutting and ultimately denaturing the invading viral genome.

Spacer Region—The spacer region is important for the invading genome detection as it encodes for a specific type of RNA called CRISPR RNA (CrRNA), this CrRNA is loaded inside a CAS protein and used as a reference, wherever the CrRNA matches the invading viral DNA the CAS protein cleave it from that place and ultimately denatures the viral DNA . The Spacer region is in fact a bacterial archive in which it stores a large number of copies of previously attacking viral DNA’s, whenever a new type of virus invade the bacterial cell the bacteria produces a special kind of CAS protein called the CAS1-CAS2 protein complex which denature the invading viral DNA and stores a copy of it in CRISPR region. This makes future detection of the invading viral DNA’s even easier. 

Palindrome Repeats—The palindrome repeats separates individual spacer DNA’s from each other in the CRISPR region. They also encodes for a special kind of RNA called the tracrRNA. This tracrRNA acts as a backbone for the CrRNA and keeps it firmly placed inside the CAS protein.

Later in 2009 Jennifer Doudna and Emmanuel Charpentier combined the CrRNA and tracrRNA in to a single GuideRNA (gRNA). They also successfully replaced the CrRNA region with a desired sequence. These discoveries lead to the foundation of use of CRISPR-CAS System for genome editing in the world of biology.

Mechanism of CRISPR-CAS System:

The CRISPR –CAS system involves 3 stages:

1.  Adaptation/Acquisition  
2.  Expression
3.  Interference  

1.Adaptation/Acquisition:

Adaptation is the starting and most important stage of the CRISPR mechanism as it keeps the CRISPR-CAS system up to date and provides a wide range of defence against a large number of viruses. Whenever a virus attacks it injects its DNA in to the bacterial cell in response to it CRISPR locus becomes active and produces a complex of two special types of CAS proteins the CAS1-CAS2 complex. This protein complex interacts with the invading viral DNA, cleaves it, produce a copy of its segment and insert it inside the CRISPR locus as spacer DNA for future use. The new coming spacer DNA is always added at the beginning of CRISPR right next to the leader sequence thus creating a sequential record of viral infection. Another protein called the integration host factor(IHF) ensures accurate insertion of the new spacer in to the CRISPR locus.

Acquisition Process 

New Spacer added in to CRISPR locus

2.Expression:

During the expression stage, in response to invasion of viral DNA, the CRISPR locus activates and transcribes the CRISPR array to from a precursor CrRNA which is then further processed in to CrRNA which is then loaded in to the endonucleases for the interference stage.

3.Interfernce:

During the interference stage in the CRISPR-CAS System, especially the CAS-9 system, the mature CrRNA is loaded inside the endonucleases molecule supported by the tracrRNA. This protein-RNA complex interacts with the viral DNA, slides over it in search of special sequences called Protospacer Adjacent Motif(PAM) which lies right next to the DNA sequence complementary to the CrRNA. This results in double confirmation of the invading viral genome and as a result the endonuclease cleaves and denatures the viral DNA preventing the viral infection.

Conclusion:

The CRISPR-CAS System has a tremendous potential of interacting with different genomic molecules and producing desired alteration in it. This revolutionary genome editing technology has opened new horizons in the field of life sciences. Scientists are now working on:

•  Gene Therapy—treating diseases by gene editing in affected cells. E.g. USA performing trials for treating HIV by knocking out genes encoding for receptor sites on T-cells.
•  Gene Drive—some genes passes faster than others in the wild, efforts are being made to cure malaria by treating certain genes with genome editing tools to make it unfit for carrying pathogen.
•  Food and livestock modification—efforts are being made to create high quality and more productive plants and animals using genome editing technology as CRISPR edited plants are escaping the GMO regulations.
•  Human Germ Line—efforts are being made to alter the genome of human sperm and egg in order to prevent spread of genetic disorders.
• Designer organisms—this technology also opens a new way for the creation of organisms which are especially designed at each growth stage and are perfect in every manner. Organisms including humans.

CRISPR- A Gift Of Nature

CRISPR- Clustered Regularly Interspaced Short Palindromic Repeats-is one of the hottest topics in discussion in the world of molecular biology and genetic engineering now days. A phenomenon once discovered to be as bacteria’s immunity against several types of phage viruses has now become the most important weapon in science’s arsenal to deal with a wide range of problems faced by the mankind. The CRISPR has become the most important tool of the genetic engineering tool box to deal with and to solve many challenges faced in the world of science. CRISPR is a molecular gene editing tools used to alter, delete and create a new genetic sequence or a new genetic combination in an organism’s genome.

History:

•  CRISPR was first discovered in 1987 by Youshizumi Ishinu at the Osaka University. He discovered these sequences in the bacterial genome when he accidently cloned a part of CRISPR sequence while working on the “iap” gene.
• Later in the 1990’s meta-genomic sequencing approach unveiled many things about this newly discovered sequence as hundreds of thousands samples were collected and tested.
• In 1993 researchers from Netherlands reported about existence of several genetic repeats in Mycobacterium tuberculosis.  
• In the same year microbiologists Francisco Mojica observed similar repeats in 2 different archaea and by 2000 his team identified these repeats in 20 different kinds of microbes.
• In 2001 Francisco Mojica and Ruud Jansen proposed the term CRISPR for these repeats. Later Rudd Jansen and his team identified cas genes.
• In 2009 the most important discovery in the world of CRISPR came the formation of guide RNA(gRNA) in a CRISPR/CAS-9 system. This was done by Jennifer Doudna (UK) and Emmanuel Charpentier (France). For the first time ever scientists were able to alter the CRISPR/CAS-9 system to their will. This discovery was like a new hope of light in the world of science, as this discovery removed many obstacles in the path of research and paved path for scientists which would ultimately lead to new discoveries.
• In 2015 an entirely new CRISPR system the CRISPR/CPF-1 was discovered which in many ways is more efficient than the CRISPR/CAS-9 system.

How it Works:

Originally CRISPR was discovered as bacteria’s natural immune system which enables them to fight and overcome the invading viral infection, whenever a viral Genome enters in to the bacterial cell the CRISPR sequence of bacterial genome is turned on, the CRISPR sequence is composed of three main components CAS gene which encodes for CAS protein (an endonuleases), Palindromic repeats which codes for TracrRNA and the Spacer DNA which codes for CrRNA, the spacer DNA itself is the copy of viral genome which attacks the bacteria, bacteria forms a copy of that viral genome and stores it so whenever that virus or a closely related virus attacks again the bacteria produces CrRNA using the viral genome copy, load it inside the CAS protein and match it with genome of invading virus if there is a match the bacterial encoded CAS protein immediately cuts and denatures the viral genome. .At first it was believed that the CRISPR/CAS-9 system cannot be used for human betterment because of its complexity but two scientists proved it wrong, Jennifer Doudna and Emmanuel Charpentier, in 2009 these two dealt with the complexity of CAS-9, First combining the TracrRNA and CrRNA to form a single Guide RNA and secondly showing that The Guide RNA can be altered according to our own will, So ultimately it can be used for betterment of mankind in many ways.

Achievements of CRISPR:

Today CRISPR is being used in many fields of life sciences to resolve ever increasing problems faced by mankind. This synthetic gene editing technology is helping us in solving many of our problems and is completely revolutionizing the world of science. The CRISPR technology enables us to cut, alter and create new genetic combinations. So far many great things have been achieved using this gene editing technology.

• Dealing with HIV is perhaps one of the biggest achievements. HIV virus causes AIDS one of the most devastating human disease, as its victim’s immune system fails to defend the body against invading pathogens. In may 2017 scientists reported in Molecular Therapy they have successfully prevented HIV infection in different model animals injected with human cells.
• Scientist reported to have created a cancer destroying genetic combination which resulted in shrinkage of cancer cells in mice which was injected with human prostate and liver cancer cells.
• Researchers have also started to alter human embryo gene in order to stop various genetic transmitted disorders. Scientists have reported to successfully remove MYBPC3 gene from a human embryo. This MYBPC3 gene is responsible for causing Hypertrophic Cardiomyopthy.
• Scientists also altered algae enabling them to produce bio-fuels twice the amount as compared to natural counterparts.
• CRISPR technology is also turning out in to a success in dealing problems with transgenic plants. Transgenic or genetically engineered plants are banned in the entire EU but CRISPR technology is proving out to be an exception as the Swedish Agriculture Board reported that CRISPR edited plant does not fall under the GMO definition as no foreign DNA is being added inside plant.
• CRISPR edited mushrooms also succeeded in escaping the USDA’S GMO regulation.
• CRISPR technology also helps in making plants resistant to various pathogens. A group of Chinese Scientist reported to have successfully developing resistance in wheat plant against powdery mildew disease using CRISPR technology.

Leading Countries in CRISPR Research:

CRISPR has probably become the most important topic to discuss and research in the field of life sciences now days, with advanced countries heavily investing in it. But some are outperforming almost everyone.

• Together USA, China, Germany, and Japan are responsible for more than 62% CRISPR publications in the world over the last five years. With USA leading the way with 1108 publications, right after the US is China with 351, Germany at third with 206 and Japan at number four with 176 publications.
• The over-all scholarly output of US alone has increased by an average 254%.
• The total number of citations received by Japanese researchers has increased by 1031%.

CONCLUSION:

                          In the end it's a simple conclusion that CRISPR/CAS-9 is a gift of nature for mankind to deal with its problems.