CRISPR/CAS technology for making better sugarcane based biofuel products

Sugarcane field

As the world continues to warm up due to excessive use of fossil fuels, climate change mitigation efforts are now greatly focused on producing such resources of energy which are energy efficient and eco-friendly at the same time. Sugarcane based liquid biofuels have been an important component of 1^st generation and 2^nd generation biofuels, however excessive demand of fuel has always been a challenge to deal. In this regard efforts focused on improving biofuel quality is of great significance.

 

The Challenge:

Sugarcane is generally rich of sugar molecules as it is a primary source of sugar based products and also traditionally have been used for the production of bioethanol for decades. A key challenge in this entire process is the “digestibility” which is the excess of various enzyme molecules to sugar content. Efforts have been made to enhance this efficiency during the stages involving production of 1^st generation and 2^nd generation biofuels, and it was only recently that with advent of modern gene editing technology a breakthrough has been achieved.

 

The Solution:

Research team from Brazil Ministry of Agriculture have been working to increase sugarcane content efficiency, the team carried out two different experiments targeting specific traits. In the first experiment the team focused on genes responsible for the production of acyl-transferases molecules and factors responsible for the rigidity of cell wall. The resultant product was the variety of cane with better access for enzyme to sugar content during hydrolysis process, which makes overall extraction of useful compounds from plant biomass more convenient. In a second related study the research group focused on enhancing the concentration of sugar content for this the silenced a gene responsible for production and relocation of sugar molecules and the silenced gene resulted in production of nearly 15% additional sucrose in stem portion while an increase of nearly 200% was observed in leaf portions. This also resulted in excessive production of glucose and fructose in it.

The resultant variety produced can result in higher quality production of bioethanol and production of better digestible products in animal feed as well creating opportunity for developing more high value addition industries in future.

Conclusion:

With annual production reaching near to 200 million metric tons better production of sugarcane and its products will be crucial for ensuring food production and climate change mitigation by increasing liquid based biofuels productions. In this view creating new varieties with modern technologies like CRISPR will be of great importance for future.

CRISPR/CAS 9 based Wood Engineering for sustainable future

 

Forests are an integral part of our ecosystem; as they play a key role in purification of environment and at the same time are a source of key products used in everyday life. But often producing these key products is injurious to our eco-system. In order to deal with such challenges research team at the VIB-Plant System Biology institute has started using modern gene editing tool for introducing desired commercial based and eco-friendly modification in to plants.

 

THE ISSUE:

Lignin is an important complex polymer present in plants and supply them with much needed structural strength, it is present in significant quantity in all plants and especially in woody plants where it is present in bark as well as the wood portion of the plant. The very compound which provides support and strength to plant also hinders its efficient processing in to useful products.

 

THE SOLUTION:

Research team at VIB institute performed an experiment few years back where the use of RNAi technology was helpful in reducing the lignin issue and increase processing efficiency in poplar tree but a key issue was negative impact on growth of plants, ultimately making it an unfavorable approach. Recently the advent of CRISPR/CAS 9 technology has changed this scenario researcher were able to introduce targeted genome modification in poplar for lignin reduction. Poplar is a diploid tree possessing two copies for each of gene responsible for a trait, the research team focused on introducing complete deletion in one copy of CCR2 gene, while introducing a mutation in to second copy, thus reducing its overall impact. The corresponding trees which have been engineered showed reduction in lignin component by up to 10% and resulted in an increase in process efficiency by more than 40%. The resulting trees were also able to grow normally indicating that no negative impact on yield has been introduced in response to genome engineering of these plants.

Poplar Wood Logs

FUTURE:

These findings can be of great influence for future research and development as the world tries to shift towards a sustainable future. As, the genetically engineered trees can be used as source of wide range of products from ordinary paper to biofuels and large number of bio-based products helping in achieving mankind’s goal of sustainable future. 

MIC-Drop: A CRISPR based technology for genetic identification and screening

Life is full of mysteries ready to be unravelled, full of questions to be answered, and full of issues ready to be resolved with sheer will, commitment and consistency, this is what exists in the realm of science and technology where researchers and powerful scientific minds work hard and fight fearlessly with the challenges of life in an effort to make the world a better place for mankind. The very passion to work and make progress has enabled world to stand a chance against the pandemic currently wreaking havoc around the globe. But challenges faced by humanity will still exist beyond the COVID-19 era and to resolve these issues will be of absolute necessity. Many of the diseases that have been around for centuries will so remain with us (including cardio vascular, neurological and other) and an imminent solution will be needed for these challenges.

In order to address these issues a group of scientist working at the University of Utah, USA has started their work on development of a new technology which enables better understanding of genetic basis of such diseases and screening of such genes resulting in to development of a cure. The researchers have used an approach that combines aspect of genetic engineering, biotechnology,  and material sciences, and is called the MIC-Drop (Multiplexed Intermixed CRISPR Droplets). The study was lead by Dr. Randall Peterson who tells that this technology will help in resolving key issues in genetic screening studies and will ultimately enable genome-sale screening.

MIC-Drop 
Courtesy: University of Utah

THE ISSUE:

Gene editing technology has been used in screening genes but the overall impact for such studies was quite limited as each of the gene would have to be treated separately with each requiring its unique Ribo-Nucliec combination making the whole

Dr. Randall Peterson, University of Utah

process exhausting and time consuming. Dr. Randall Peterson said:

“The process has always been focused on a single gene or a single modification at a time So if you want to do 100 genes, it's 100 times as much work”.

 

THE SOLUTION:

In order to deal with the issue the Team of Dr. Randall Peterson came up with an innovative idea of MIC-Drop technology, in this the team first prepared a large number of gRNA’s and protein molecules followed by packaging of these components in to micro-droplets and each of the droplet was given a unique identity inform of a DNA barcode. The chemical properties of droplets was engineered in order to make them stable and prevent any intermingle of their particular material contents while delivering in to host cell systems. The drops are delivered in to separate embryo cells using specific microscopic needle and the development for each of the cell is monitored later on.

THE SUCCESS STORY:

In order to demonstrate the potential of this tech the team worked in collaboration with researchers from Harvard Medical School and Massachusetts General Hospital, the work was focused on testing about 188 genes in zebra fish associated with heart development, for the experiment each embryo cell was loaded with its unique CRISPR containing drop, many of the resulting fish developed some sort of heart issue and a total of 13 genes were screened out associated with this issue. Indicating towards many aspect of heart development which were previously unknown.

Dr. Saba Parvez said:

“Previously, setting up a CRISPR screen of hundreds of genes in zebra fish would have taken a team of researchers many days and required hundreds of needles, Now you have streamlined that process into one user doing it in a span of a couple of hours,”

 

CRISPR a ray of hope to cure Pain forever…

 

Pain is the most unpleasant type of sensory response received by human brain and is often more or less associated with an under lying condition which tends to inflict some sort of physical damage to body tissues, the very sensation pushes human conscience to develop strategy to protect its damage body parts for enabling proper healing and recovering full working capacity of damaged tissues. But this very sensation has a darker aspect, in certain diseases pain to be endured is too much for any human being and this often leads to decision relating to ending one’s life. Another key issue exists in form of chronic pain where a person consistently feels pain for months or even years despite of recovering from original cause in form of an injury or a disease, according to an estimate ranging from 19-50% of global population experiences chronic pain for a greater part of their life and in response recommended opioids often produce numerous side effects which leads to development of some diseases or may cause conditions of substance abuse. Now this overall situation demands a solution one that can suppress or cure pain in certain medical cases and prolongs life span with a possibility of some cure in near future. In this regard the CRISPR based genome editing is now proving to be a ray of hope at the very end of a dark tunnel.

The pain tunnel

Now the brain experiences sensation of pain upon receiving some sort of stimulus like touching a hot pan, getting some sort of cut and so on, this triggers a series of electrical signal inside human nervous system which convey signals of pain. Inside body the nervous system is made up of a neurons and each neuron has small pore like openings present on its surface called the ion channels. These channels consistently open and close to allow neuro-chemical communication in nervous system and among these channels is the sodium Na v1.7 channel which allows pain signalling with influx of sodium ions inside neurons. Although there are many studies which have suggested that a sodium channel called Na v1.7 could play a central part in chronic pain, but in various cases when people have mutations in the gene coding for this channel, they either experience extreme, constant pain, or can’t feel any pain at all. Scientist have been trying to block these Na v1.7 channels by using certain small-molecule drugs and antibodies, but they often struggled because these therapies also interact with structurally similar sodium channels in the body leading to severe complications, in order to deal with this issue and find a cure for this pain by manipulating ion channels researcher from University of California, San Diego lead by PhD candidate Ana Moreno decided to harness power of modified version of CRISPR/CAS9  molecule which tends to locate gene with precision and modify its function without any off target effects.

Pain signals are transmitted to the brain through neurons similar to these in the spinal cord

CRISPR Ray of Hope:

the team utilized a variant of CAS9 called the dcas9 (de natured CAS9)  in which the molecule perform all standard functions from gene targeting to binding but only the cleavage function is absent, the molecule is then attached with a repressor molecule which prevent expression of that gene, the team encoded this dCAS9 for Na v1.7 gene in order to suppress its expression and reduce pain sensation. Molecule was then injected in to a mice followed by chronic pain stimulus with chemotherapy and results show high tolerance of pain in mice for up to 44 weeks, similar level of tolerance was also observed in mice that previously had issues involving chronic pain. The dCAS9 molecule only binds with certain gene and prevent its expression for a certain time this also prevents permanent changes in DNA and revives the ability to feel pain once the baseline cause of chronic pain has been resolved bringing back life to normal.

Senior neuro-scientist Sulayman Dib-Hajj, at Yale University describe that although this is still far from human trials but it is a great  and highly successful start with nearly 44 week relief this can revolutionize the entire treatment procedures for various painful diseases.

Conclusion:

Cure of pain for a certain time frame can provide various patients a temporary necessary relief from serious pain experienced during treatment procedure like for cancer treatments and so on. Also it can provide number of options for athletes to soldiers as for how they really want to deal with their injury and what level of compromise they would be ready to make with their profession for healing a certain injury. This research also provide a possible hope for also recreating pain sensation by certain degree of reengineering in case of certain disease related to pain insensitivity which often lead to serious life threatening injury and loss of organs in patients having such conditions.

CRISPR for curing Muscular Dystrophy

Human body is composed of a complex mixture of muscles responsible for a series of functions to ensure smooth running of body. Muscles are responsible for the movement of bones and skeletal structures, enable organ movements and functioning i.e; movements of digestive tract and pumping function of heart and enables many sorts of hormonal regulations inside the body. Error in the muscular system can lead to a series of degrading effects causing not only damage to the organ systems, but also resulting in to dysfunctional skeletal system rendering a person incapable of movements. Many genetic disorders results into such collapse of muscular functioning over the ages, one of such disease is the myotonic-muscular dystrophy.

 

THE CAUSE:

The disease is genetic in nature and can be easily transmitted from one generation to the next, in addition to this the disease is autosomal-dominant type and there are two types of muscular myotonic disorders caused by mutations in two separate genes; the Type-I and Type-II with type-I being ore fatal. The type-I is caused by mutations in a gene named “DMPK” which encode for a specific protein kinases involved in impulse transfer and communications in between cells. The mutation in the gene produces an unstable region inside it producing longer than normal RNA’s in cell, these RNA’s began to trap certain essential protein molecules in cells disrupting normal functional biochemical chain and resultant protein also becomes toxic in nature. This causes a breakup in communication and functioning system of cells and muscles remain contracted for long time resulting in muscle deterioration. Onset of the disease is during the middle ages of a person and in severe cases causes a reduced muscle mass in lower legs, shoulders, neck and face, also it results in clouding of retinal muscle and errors in cardiac conductions. Often people develop respiratory disorders as well due to poor muscular functioning as result of this condition.

Patient with Myotonic-Dystrophy

 THE CURE:

So far there is no effective way to prevent this disease and no proper medication to reduce the chances of disease onset, only treatment so far involves a balance diet and regular physiotherapy of patient. A possible cure for diseases like this was a fantasy for a long time until now.

A group of researchers lead by Dr. Gene Yeo at the UC San Diego, California has conducted two different studies in an effort to cure such disease; the first study was conducted in 2016-17, in this study the team took normal CRISPR/CAS9 molecule and complex reverse engineering prototype molecule was created designed to cleave target RNA instead of traditional CAS9 molecule whose cleave target is DNA. Then the prototype molecule was tested on a cell culture isolated from muscular dystrophy patient and the degrading acclivity of newly formed RCas9 molecule was measured. Initial results were highly convincing as the new molecule neutralized up to 95% of the toxic RNA molecules in cell. In second study published in 2020 the novel RCas9 molecule was tested for its efficiency in mouse model of myotonic dystrophy disease. The result showed that a dose of RCas9 molecule massively reduced the accumulation of these toxic RNA molecules inside mouse body cells thus reversing their deleterious effects and resulting in to gradual increase in body muscle mass and stable functioning of the body. On successful trial for this technique Dr. Yeo said:

“There are no cures for such muscular diseases, this study opens up a new gate towards the treatment and cure of this and at least 20 other such hereditary muscular diseases like the ALS.”

Dr. Gene Yeo

Conclusion:

Such new study are consistently opening new doorways towards the possibility of multi-dimensional usage of a technology which remained with humans for century and was left unnoticed until a decade ago and it tells how much can mankind gain from the immense revolutionary potential of this technology.

Curing Blood Disorders using CRISPR

Blood is a fluid or a semi-liquid material that flows within the human body, circulating around from our head to our toes and is crucial for the survival of humans. Blood is composed of a diverse a unique mixture of cells and biochemical molecules and each one performs a specific function which is essential to the survival of a human. These blood related molecules are crucial for multiple life sustaining activities like the transportation of oxygen O₂, healing and repairing of wounds, translocation of sugar molecules etc. Some of these molecules (cells) are often unable to perform their function because of certain genetic mutations resulting in to malformation of cells and ultimate reduction in activity of those cells which threatens the survival of a human.

THE DISEASE:

Sickle cell and thalassemia are among the most common blood disorders and are cause d due to a specific genetic mutation in genes encoding for haemoglobin (molecule transporting oxygen). According to a detail study published in LANCET in 2015-16 a total 4.5 million peoples are affected by the sickle cell where as more than 228000 people are affected by thalassemia disease and annual deaths cuased by the 2 diseases is around 130,000 annual. This implicates the severity of this disease all over the world, in addition to this patients who continue to live with these disorders are faced with severe problems including repeated painful blood-transfusion, feeling exhausted, weak, pale skin colour, oxygen deprivation and more.

OLD SOLUTIONS:

Treatment of this disease often involves severe blood transfusions, intensive care, medicines and in severe cases stem cell therapy which is often faced with multiple challenges including scarcity of donors, rare donor cross match, and chances of graft rejection, which results in severe outcomes.

NEW SOLUTION:

Owing to the risk and rare possibility of success related to previous treatments need for modern approaches is inevitable, one the most prominent approach for curing genetic disorders is the use of CRISPR-gene editing technique. A detailed study is being performed by CRISPR-Therapeutics and US pharmaceutical giant Vertex Pharmaceuticals in US, the study involves the application of a detailed process using gene editing called the “CTX001” which makes some specific point cuts in to the DNA for the alteration of certain genes and triggering haemoglobin production.

THE PROCESS:

The study is currently being carried out at the Sarah Cannon Research Institute in the US state of Tennessee where many patients are currently enrolled for the trial. One of the very first patient enrolled in the trial has recently being evaluated for possible improvements in health conditions and the results are astonishing.

The trial itself involves using the CRISPR gene editing process to clip of certain “SWITHC-OFF” genes present in the host stem cells responsible for the turning off the production of fetal haemoglobin or the HbF resulting in to the production of fetal-haemoglobin molecules. The HbF  itself is a kind of haemoglobin molecule that exists in the body during fetal stage and is later replaced by the adult version of haemoglobin but has almost similar tendency of carrying out all normal functions.

THE RESULT:

Recently results for the initial phase of the research were released by the team which states that the blood analysis of Victoria Gray (the 1^st patient) an approximate 47% of stable fetal haemoglobin (HbF) in the blood with nearly 95% of them carrying the edited version which is beyond expectations and a huge success.

Dr. Jeffrey Leiden, Chairman and CEO of the Vertex Pharmaceutical said:

“The result are remarkable and shows the ability and effectiveness of CTX001 for curing these severe blood disorders. With this great initial phase success we are looking forward to work along with researchers, doctors, patients and thier families in a close relationship in future, and we hope to soon expand the treatment impact to cure other serious diseases such as the Duchenne muscular dystrophy and myotonic dystrophy type 1.”

Dr. Jeffery Leiden, CEO Vertex Pharmaceuticals 

CONCLUSION:

The technology is proving itself to be a highly effective method to resolve many lethal diseases which have been a major challenge for decades, after promising results from animal trials to successful clinical trials the tech is now almost ready for broader application, but the most necessary thing here is to get the public ready for it and smooth, swift policy regulations by the regulatory authorities and legislators to ensure in time arrival and application of the technique and way before even this tech began to be obsolete for rising challenges.

CRISPR-Chip: Detecting Mutations from Raw DNA

Since its introduction to the world CRISPR technology has revolutionized every aspect of life and is creating a huge impact in every man’s life. The tech enables the editing of genes but with more focused research and technical advancements the tech is being used for creation of more sophisticated devices, in a recent efforts a team of researchers working at the UC Berkley, USA have recently developed a chip based device using CRISPR molecules for the detection of mutations from unamplified DNA samples. The team is headed by Dr. Kiana Aran who specialises in bio-medical engineering.

Need for the technology:

Genome sequencing and detection of anomalies inside the DNA are becoming prominent for accurate diagnosis and treatment of the disease. This technique of presice medicine has developed to a greater extent in past half century, but still requires some expensive machines, reagents, and trained workforce for proper functioning. Also it requires an additional imaging device for visualizing the results. All this is a set-back and consumes both money and time. So, new techniques regarding these kinds of diagnosis are required.  

Development of Chip:

In an effort to answer the problem Dr. Kiana Aran and her team at UC Berkeley developed a chip based diagnostic device. The device consists of two key components:

• The dRNP- CRISPR molecule
• Graphene based field effector transistor (gEFT)

The chip is formed by combining the two components, the CRISPR-CAS is a DNA cutting tool having the ability to detect specific DNA segment with extreme precision, for the detection chip module a slightly modified module of CRISPR is being used which is able to detect the specific DNA segment but its ability of cutting the DNA has been deactivated. These modified CRISPR molecules are stacked on top of the graphene transistor layer, the graphene layer was preferred over the silicon based layer due to its higher electric sensitivity than the silicon.
What is graphene? See: https://crisprpedia.blogspot.com/2019/09/graphene-magic-material.html

CRISPR-gEFT Chip module

Functioning of CRISPR-Chip:

The raw DNA sample without subjecting to any amplification process is loaded on to the chip set. The DNA is taken by the CRISPR molecules and unzipped, it analyzes the whole segment of the DNA until it reaches hat part of the DNA where a specific part of the DNA or gene is present that has complementary sequence to the guide RNA present inside the CRISPR molecules. When the specific part is identified it binds with it and this hybridization of target DNA with the CRISPR module influence the electrical characteristics of graphene surface. This produces a signal resulting detection of mutations and abnormalities in DNA in short period of time.

Initial Trial:

For initial validation of the device a trial was conducted to check the effectiveness of the tech for this Duchenne muscular dystrophy (DMD) disease was selected. This disease is caused by a exon deletion in the gene resulting in production of dysfunctional dystrophin resulting in to malformation of muscle fibres. Two of these exons the exon-3 and exon-51 are absent in all DMD patients. For the trial CRISPR chips specific for exon 3 and 51 were designed and DNA samples from the DMD patients and healthy donors were analysed using chip. Results exhibit that the healthy sample with exon 3 and 51 produces a strong signal in comparison to the DMD patient DNA sample.

Published Results-Courtesy Nature

Conclusion:

The device is an effective way for detecting certain mutation based disease on early stages, enabling on time detection and better diagnosis and treatment of the disease. The tech is a promising and effective one ensuring further enhancement in precision medicines.

Courtesy: Nature, UC Berkeley, Aran Lab.