CRISPR is a genome editing tool that is creating a buzz in the science world. CRISPR has been the most discussed topic in the scientific community since the end of the 20th century. It is simple, faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.There are consistent predictions that this gene-editing technology has the potential to transform our planet. Researcher uses this technology to easily alter DNA sequences and modify the gene function. It has many potential applications including correction of genetic defects, treating and preventing the spread of diseases and improving crops.

CRISPR-Cas9 as gene editing tool:

CRISPR is an acronym for “Clustered Regularly Interspaced Short Palindromic Repeats.” CRISPR genome engineering technology enables scientists to easily and precisely edit the DNA of any genome. "CRISPR" (pronounced as crisper) is shorthand for "CRISPR-Cas9" CRISPRs are specialized stretches of DNA.

In nature, CRISPR palindromic repeats play an important role in microbial immunity. When a virus infects a microbial cell, the microbe employs a special CRISPR-associated nuclease (Cas9) to chop off a piece of the viral DNA. The nuclease is directed to its target sequence by a short RNA fragment called as a guide RNA (gRNA), which is complementary to the target segment of the viral genome. The snipped DNA fragment may then be stored between the palindromic CRISPR sequences to retain a genetic memory for disabling future infections from the same viral strain. Once scientists learned how the CRISPR system worked in bacteria, they figured out how to reprogram it to allow efficient editing in any species. This revolutionary breakthrough has completely change the way science is performed, and it will soon change many aspects of our everyday lives.

CRISPR is a specialized region of DNA with two distinct characteristics: (i) the presence of nucleotide repeats and (ii) spacers. Repeated sequences of nucleotides- the building blocks of DNA are distributed throughout a CRISPR region. Spacers are bits of DNA that are interspersed among these repeated sequences. The protein Cas9 (or "CRISPR-associated") is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA.CRISPR-Cas9 is cheap, easy to use, precise, and remarkably powerful tool as compared to older and other popular tools used for genetic engineering. There are wide range of applications of CRISPR from human biology, agriculture, and microbiology.Researchers are still trying their best to figure out how CRISPR can be used to makebetter world, though the power to alter DNA brings many ethical questions and concerns.

Latest Trends on CRISPR Research:

1. To treat Genetic Errors by using CRISPR:

            Hypertrophic cardiomyopathy (HCM) is a heart condition that affects roughly 1 in every 500 people worldwide. Its symptoms are painful and often deadly. Mutations in a number of dominant genes cause the heart tissues to stiffen, which can lead to chest pain, weakness. In severe cases, sudden cardiac arrest may also occur. Still, there is a hope that in the due course of time, we will be able to use gene editing to cure this disease once and for all.In 2017, scientists at the Oregon Health and Science University used CRISPR to delete one of these defective genes in a number of viable human embryos. The results were promising: Of the 54 embryos that were injected with the CRISPR-Cas9 machinery after 18 hours of fertilization, 36 did not show any mutations in the gene (practically no chance of developing the disease) and 13 were partially free of mutations (with a 50 percent chance of inheriting HCM). On further reducing the chances of inheritance, it was found that only one was a mosaic.

2. Improving efficacy through algorithm based approach:

            As per the report published in November 2018 in the leading journal Nature Biotechnology, CRISPR-Cas9 will enable scientists to predict the best sequences as a target to make gene editing more reliable, cheaper, less time consuming and more efficient. In this attempt, the researchers analyzed over 40,000 pairs of different target DNA and guide RNA deeply, and carried out CRISPR-Cas9 gene editing in different cells. It was analyzed that the repair depended on the exact sequence of DNA and guide and discovered that it was reproducible within the same sequence. Thereafter, a machine learning computational tool, called FORECasT was generated, that enabled them to predict the repaired sequence, using the targeted DNA sequence alone.

 

A machine-learning algorithm in Delphi has been created in a similar attempt that predicts how human and mouse cells respond to CRISPR-induced breaks in DNA. It was discovered that cells themselves repair broken genes in precise and inevitable manners, typically even returning mutated genes back to their healthy version. In fact, the researchers were able to successfully correct mutations in cells taken from patients with one of two rare genetic disorders.

3. Elimination of the pathogens via CRISPR Cas9:

Even after 38 years of discovery of HIV, scientists still haven’t found a cure. CRISPR can turn out to be a hopeful measure in this regard. In 2017, an experiment was carried out on mice to increase resistance to HIV. A team of Chinese researchers successfully implemented this by replicating a mutation of a gene that effectively prevents the virus from entering cells. This mutation has been found to occur naturally in a small percentage of people. It can be induced artificially using CRISPR, which will considerably bolster HIV resistance in humans in the future. In another approach, scientists from North Carolina used CRISPR technology to engineer bacteriophages, to develop a proven, safe method for treating harmful bacteria.

4. Recreation of species using CRISPR:

George Church, a Harvard geneticist and his team have invested their valuable 11 years to recreate the DNA blueprint of the extinct mammoth. DNA from mammoths preserved in Arctic permafrost has been used to discover that only 44 genes are separating mammoths from elephants.It is an attempt to save endangered elephants they are trying to create hardier mammoth hybrids that is much more cold-resistant. The researchers also look forward to inserting non-mammoth genes, which will prevent them from growing tusks, to prevent poaching, and new DNA to allow them to eat a wider diet.

5. CRISPR could create healthier foods:

CRISPR gene editing has proven to be promising in the field of agricultural research. Mushrooms that do not turn brown have already been created using CRISPR technology. Few attempts have been made by Scientists from Cold Spring Harbor Laboratory in New York used the tool to increase the yield of tomato plants. The lab developed a method to edit the genes that determine tomato size, branching architecture and, ultimately, shape of the plant for a greater harvest.The leading company, DuPont Pioneer is trying to bring its “waxy” gene-edited corn into the U.S. market by 2020. The U.S. Department of Agriculture regulation has already approved gene-edited mushroom, since it doesn’t contain genomes from viruses or bacteria, declaring it the first CRISPR-edited organism to be green-lit. Before that, we should know the difference between gene-edited crops and GM crops. While traditional GMOs are made by inserting foreign DNA sequences into a crop’s genome, makes precise alterations to genes in specific locations of the native genome.

6. Eradication of Mosquitoes:

Gene-editing techniques like CRISPR could directly combat infectious diseases, but some researchers have decided to slow the spread of disease by eliminating its means of transmission. Scientists at the University of California, Riverside have disrupted target genes in multiple locations of the mosquito’s genes, using gene drive technology, by impairing the mosquito’s flight and vision. Success was achieved in postponing mosquito development, shortening the animal’s lifespan, retarding egg development, and diminishing fat accumulation. Other researchers at the Imperial College London, have tried another unique way to get rid of a female mosquito, by interfering with how they reproduce.

Current approaches in arthropods are based on delivering the gene-editing Cas9 on to eggs by embryonic microinjection, a tough and inefficient method that works in a mere a tiny low range of species. As per Jason Rasgon, faculty member of zoological science and medicine, Penn State school of Agricultural Sciences, microinjection will harm the eggs, and it needs costly instrumentality and coaching to implement.

To address these limitations, the researchers developed ReMOT Control—Receptor-Mediated Ovary Transduction of Cargo. This is the technique that will deliver a Cas9 system to the target by a simple injection into the blood of feminine arthropods, wherever it is often introduced into the developing eggs via receptors within the ovary. Rasgon explained that in ovary and egg maturation, mosquitoes and alternative arthropods synthesize food proteins, that are secreted into the blood and brought up into the ovaries. The assumption is to bypass the requirement for embryonic microinjection to attain ordering gene editing within the embryo.

7. Designer babies

In 2015, the first edit in the genes of a human embryo in vitro was made by China using CRISPR + IVF, which sparked global protest and pleas not to make a baby using the technology. As per the reports, a team at the Southern University of Science and Technology lead by HeJiankui had been recruiting couples so as to create the designer babies. They had planned to eliminate a gene called CCR5 hoping to make the offspring resistant to HIV, smallpox, and cholera. They have successfully brought two healthy, little Chinese girls named Lulu and Nana into the globe in November 2018. He Jiankui said that his team performed “gene surgery” on embryos created from their parents’ sperm cell and eggs to shield the youngsters from the human immunological disorder virus, HIV, that causes AIDS. Their father is HIV-positive.

Tips to build a successful career in CRISPR technology Field:

            Indian Research Community is all set to adapt for this trending gene editing technology of CRISPR Cas9. In fact, a lot of research is already being conducted at Govtresearch labs and private biopharma companies with respect to CRISPR Research Trends to tackle issues faced globally related to health, agriculture and Food technology.

List of Research Institutes in India where CRISPR research is being conducted:

• IARI – Indian Agricultural Research Institute (IARI)
• NIPGR – National Institute of Plant Genome Research
• JNU – Jawaharlal Nehru University
• DU – Delhi University
• NBRI – National Botanical Research Institute
• NABI – National Agri-Food Biotechnology Institute
• JUIT – Jaypee University of Information Technology
• ILS – Institute of Life Sciences, BBSR
• NII – National Institute of Immunology
• NBRC – National Brain Research Centre
• IIT – Indian Institute of technology – All Locations
• IIsc – Indian Institute of Science, Bengaluru
• NRCPB – National Research Centre on Plant Biotechnology
• CSIR-NEIST – CSIR North East Institute of Science and Technology
• CSIR-IICB – CSIR Indian Institute of Chemical Biology
• CSIR NCL – CSIR-National Chemical Laboratory
• TIFR – Tata Institute of Fundamental Research
• NCBS – National Center For Biological Sciences
• CCMB – Centre for Cellular & Molecular Biology

List of Biopharma companies in India Where CRISPR research is being conducted:

• Reliance LifeScience
• Thermo Fisher
• Bayer
• Syngene
• Merck
• GE Healthcare

Job opportunities related to CRISPR technology:

There are many job opportunities in this field for which you need to apply for vacancies in CRISPR related research projects at either research institutes or Biotech companies. But all of these jobs require a minimum qualification of being a postgraduate with NET / GATE qualification if you are joining a research lab or preferably a PhD degree (given priority).

• So, the first step here is to grab a PhD position for yourself after clearing any of the exams that can help you get admission as well as guarantee you a fellowship. E.g. CSIR-JRF, ICMR-JRF, etc.
• You need to select for a potential guide, who himself is well aware of this new technique and can provide you with necessary facilities and guidance.
• Next, one should develop a research problem which has novel applications, that includes the use of CRISPR and which has not been standardized properly. But always keep in mind that you require something to look upon, when in doubt. So, try to select a research problem, which may have been successfully implemented in some other way in some related model organism.
• Now, you should also have a basic understanding of genetic engineering and molecular biology protocols as well. E.g. like cloning, PCR, qPCR, cDNA synthesis, RNA extraction, immunochemistry techniques like western blotting, immunofluorescence. If there is no possibility to keep all these techniques in your research work, you can always opt for hands-on training courses or workshops which can be availed from any of the reputed laboratories.
• It may require additional hand-on-expertise on mammalian cell culture- experience handling multiple cell lines, transfection, electroporation, transduction, generation of stable cell lines, single cell clonal selection, RNAi mediated gene knockdown, transient and stable expression of proteins in mammalian cells.
• Again, in order to get experience in related techniques or CRISPR itself, you can join as a Junior Research Fellow in reputed laboratories.
• Once you have completed your M. Sc./ Ph.D. research work successfully, you can apply for a patent as well, along with the publications in reputed journals. These publications will give you an upper hand while applying for the jobs.
• Moreover, you can apply for Postdoctoral positions in similar fields, as limited knowledge may give you perfection in a particular field, but it will not give you exposure. The more you learn, the better are the chances for you to get a salary hike or promotion or possibly a government job as a scientist.

CRISPR Tomorrow:

Let me remind you that CRISPR will offer you highly paid jobs.Since his field is still in its infancy, you will get ample opportunities to prove yourself. Current scientific advancements have proven that CRISPR is not only an extremely versatile technology, it’s proving to be precise and increasingly safe to use. But a lot of progress still has to be made; we are only in the beginning to see the full potential of genome-editing tools like CRISPR-Cas9. Till date, we are facing technological and ethical hurdles that are not allowing us to feed the hunger-stricken planet, remove genetic disorders, or conserve extinct animal species back to life. But we are making progress in a satisfactory manner. But one thing is obvious that CRISPR is a technology which in near future will be adapted globally looking to its diverse applications but of course, the practice should be done under strict guidelines to avoid its misuse.