Construction and testing of CRISPR-Cas9 constructs for the resistance of cassava to cassava mosaic viruses

Cassava (Manihot esculenta Crantz) has been used as a staple food in many nations. As of 2016, over 800 million people depended on cassava as their staple food. No doubt, cassava is amongst the most important crops in Africa because it is drought tolerance and produces high starch. However, cassava mosaic virus is known to cause unbearable yield loss in cassava starch. Every year, millions of US dollars are spent in central and east Africa trying to manage, prevent and control the spread of the cassava mosaic virus.

My idea for sustainable crop protection involves the use of molecular tools to find unknown strains of cassava mosaic virus which are affecting cassava in sub-Saharan region, particularly in Zimbabwe, and use CRISPR-Cas9 gene manipulating tool to create a viral resistant cassava plant. CRISPR, together with CRISPR associated endonucleases (Cas9), function as an adaptive immune system for bacteria and archea. In bacteria and archea, CRISPR guide the Cas9 endonuclease to target specific nucleic acids of bacteriophages based on sequence complementarity and cas9 will induce a double strand break on the viral DNA, rendering it ineffective. The objective of our (me and my supervisor) project is to try and imitate the same CRISPR-Cas9 process in plants for crop protection against invading virus.

Specific aims of the project involves

  1. The extraction and sequencing of DNA from infected cassava leaves
  2. Use these sequences to create a range of CRISPR/Cas9-sgRNA constructs which targets very important regions on cassava mosaic virus DNA, regions such as the origin of replication, Rep gene and movement protein gene, to induce double strand breaks on CMV DNA
  3. Transiently express these constructs in Nicotiana benthamiana (our model plant) which is infected with cassava mosaic virus
  4. Determine the effect of the CRISPR/Cas9 constructs on the replicating virus in infected plants (using quantitative PCR).

If these constructs are put in a cassava mosaic virus-infected plant, they should chop up the cassava mosaic virus DNA in the plant and mutate it, hence the plant will bloom and grow because the viral genes will have been made non-functional. As a result, high starch content will be obtained from these resistant plants. If the project is successful, farmers will enjoy the benefit of growing crops in virus infected areas since the presence of the virus will not do anything to these resistant plants.

This project will have an economic impact in the sense that our farmers will no longer be needing pesticides or insecticides to kill viral hosts (Bemisia tabaci) since the presence of the virus will do nothing to the plants. This will save money and hence increase farmer`s profits. This technique, if used by other scientist, can improve crop protection of many important crops such as maize and rice. Another advantage is that since no chemicals will be used to spray the plants trying to control cassava mosaic disease, land pollution by chemicals will be greatly reduced so we can keep our environment clean and friendly.

So far we have made big progress in this project. We have managed to extract DNA from infected plants and sequenced it using a minION from Oxford Nanopore technologies. Sequenced viral DNA was used to create a range of guide RNA for CRISPR-Cas9 constructs which targets hairpin, intergenic region, Rep protein gene and Rep enhancer gene on DNA A of cassava mosaic virus. As I write this document, we have sent these guide RNA for synthesis in South Africa.  We have also planted plants in our Lab which are waiting to be inoculated with the virus.

This project will have a very big positive social impact on the general population. Also, if we manage to protect cassava plants from cassava mosaic viruses, we can eradicate malnutrition in underdeveloped and developing countries because high starch content will be obtained per unit area with these resistant plants.

If there is anything I am passionate about, is to see the world happy. No doubt, one of the biggest source of happiness for every parent or any president in this world is to be able to put enough food on the table. But because of these plant viruses, we face many challenges in crop protection every time. What motivates me to do this project is that feeling of knowing that I am playing my role as a citizen of this world to provide healthy plants to the community for better health of other citizens. With my position as a scientist, protecting crops from viruses will result in increase in yield. Africa is failing to produce enough food for its citizens. One of the reason being the failure of farmers to protect their plants from viruses. This reason has motivated me to be that one person standing in place of farmers to protect their crops from viruses. Of course, I want to be an N-GAGE winner, so that I can continue pursuing this project and be able to network with other expects in this field of molecular biotechnology to obtain valuable ideas in crop protection. You can also check the other program for plant protection I attended on this link https://www.youtube.com/watch?v=_o64Sq5naeI&t=80s

Tapiwa Nyakauru, Zimbabwe

65 comments

  1. Brilliant presentation of noble intentions. Just one note … it is going to be GM-cassava variety, because you need that DNA construct to be present in every cell of the plant and to be expressed to fight with virus DNA. And as alternative to CRISPR, The Plan B, I would like to suggest more classical, traditional approach to creation of viral-resistant plants. Inhibitory small RNAs. It can be your back-up if CRISPR fails to deliver stable reproducible results.

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