Work done by: Shireen S1, Vaishnavi Mukkawar1, Rohit Bhatia2, Taruna Arora3
1: Department of Biotechnology, Jamia Millia Islamia, Delhi, India
2: Division of Sustainable Technology, Rudraksh Prodhyogiki Sangathan, Delhi, India
3: Department of Biochemistry, Institute of Home Economics, University of Delhi
Introduction
Bacterial cell protects itself from invading viruses by stealing sequence stretches from the invading virus’s genetic material and incorporating into its own. These newly incorporated sequences are called CRISPR i.e. Clustered Regularly Interspaced Short Palindromic Repeats. The bacteria then make RNA copies of this sequence called CRISPR RNA (crRNA) which acts as a guide for a CRISPR associated genes such as Cas9 which is a nuclease. A nuclease is like a scissor that cuts DNA. Scientists, with a few modifications, use this system as a genome editing tool. By replacing bacterial CRISPR RNA with a modified guide RNA, scientists can control the location at which cas9 will cut the DNA. The enzyme cas9 scans the DNA for the sequence complementary to that of the guide sequence and chops off the DNA upon encountering the sequence thus destroying it. With certain modifications in the system, scientists are able to cut a particular stretch of DNA and replace it with another one, using cell’s own repair mechanism called homology-directed repair or silence a gene i.e. shut it down.
CRISPR was an accidental discovery from Osaka University researcher Yoshizumi Ishino in 1987. The function of these sequences was identified by Francisco Mojica at University of Alicante, Spain. The modifications that allowed CRISPR-Cas9 system to be used in a variety of ways was carried out by Jennifer Duodna and Emmanuelle Charpentier in 2012 at University of California, Berkeley.
Due to the ease and extent of manipulation provided by this technique, its applications are growing very rapidly. It has already been used to modify genes of plants, insects, and animals.
Applications of CRISPR-Cas9
Plants: Due to ever increasing world population and climate change there is urgent need to develop plant varieties with desired qualities, for e.g. higher yield or pest resistance. The CRISPR-Cas9 system provides a one step method to obtain the perfect plant without the interference of multiple undesired traits that were obtained (along with the desired trait) by multiple breeding cycles. This technique provides a very high level of precision for introducing the desired gene into a plant. Also, a part of the plant’s genome can be deleted in order to obtain desired qualities for e.g. in mushrooms, a specific enzyme that causes browning of the mushroom was deleted in order to obtain an anti-browning mushroom. As a result, the shelf life, i.e. the duration for which it is fit for consumption, of CRISPR-edited mushroom, increased, it resisted blemished that arose while handling the mushroom and DNA from any other organism was not introduced into its genome. Thus CRISPR-Cas system allows us to make changes in the genome of an organism without involving any foreign DNA.
Insects: The vector borne diseases are responsible for over 1 million death globally. The insecticides used for vector control are harmful to the environment and also cause number health problem to humans. These drawbacks necessitate the development of methods which can effectively control vectors without harming environment and humans. The CRISPR-Cas9 has emerged as a technique fulfilling both these criteria. For example, CRISPR-Cas9 has been used to produce genetically modified mosquitoes that do not transmit malaria. Scientists have shut down the genes necessary for female Anopheles mosquitoes for making eggs and the mosquitoes are genetically engineered to spread this trait quickly. This means once the genetically engineered mosquitoes are out there, the trait will spread very quickly and the female population of Anopheles mosquitoes will be sterile, giving us a chance at wiping malaria. Similarly, CRISPR is also being used to prevent transmission of Dengue, Zika, etc.
Animals: Gene editing of animals lik a pig are being carried out to make its organs more suitable for transplantation in humans. This technique can be used for genome editing in live cells, which makes it even more useful and important. It can be used to target genetic diseases like Huntington’s disease, cystic fibrosis etc. The defective copy of the gene responsible for the disease can be replaced by the functional copy of that gene. It has already been to cure sickle cell anemia and the treatment may be available as an effective therapy in a few years. CRISPR-Cas9 system can also be used to study and hopefully combat diseases involving multiple and complex genetic alterations like that in cancer. Extensive research is going on for this purpose. China has even used CRISPR edited cancer fighting white blood cells to fight metastatic lung cancer in a live human being.
The CRISPR technology is developing by the day. Gene editing is easy and efficient when done in an embryo or cultured cells, but it becomes a lot more complex when it comes to editing in adults. These issues once resolved could be of extreme usefulness in the development of such techniques. CRISPR-Cas9 system, discovered just nearly 5 years ago, has a bright future in combating many diseases and in the production of transgenic organisms. Considering the range of manipulations it allows, from silencing a gene to replacing a defective gene copy with a correct one, it is indeed a very versatile technology having a plethora of applications.