Cocoa is grown across Africa, Asia and the Americas in about 57 countries on an estimated total area of 6.5 million hectares. Crop losses caused by pest and disease are the major production constraint in all cocoa producing countries with estimated losses ranged between 30 to 40% of global production. Black pod of cocoa, caused by different Phytophthora spp., is one of the most destructive diseases in almost all countries where this crop is grown. So far, there are no effective chemical, biological and cultural measures to control black pod disease of cocoa. Therefore, due to the lack of resistant cultivars and ineffective control strategies, the development of genetically improved Phytophthora-resistant cultivars is the only available option and should be taken up as a top priority. Recent studies indicated that expression of targeted genes is increased when cocoa plants are infected by Phytophthora. Of which, targeted genes are highly expressed, suggesting that these genes may play a role in modulating the response to fungal infections in cocoa, and knockout of these targeted genes may lead to Phytophthora infection resistance.
The aim of the proposed project is to develop Phytophthora-resistant cocoa variety via CRISPR/Cas9-targeted knockout of the potential susceptible genes. It is hypothesized that the simultaneous editing of the potential susceptible genes via CRISPR/Cas9 system enhances Phytophthora-resistant in cocoa. CRISPR/Cas9 is easier, more cost-effective, precise and is highly efficient even at multiplex genome editing in comparison of other genome editing tools such as zinc finger nucleases (ZFNs) and transcriptional activatorlike effector nucleases (TALENs). Sequence-specific nucleases (SSNs) have played an immense role in crop improvement and are among the cutting-edge technologies, of which the combination of clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 nuclease protein is the most effective SSN used in various crops. In several previous reports, CRISPR/Cas9 system has been shown as a useful strategy for the management of several diseases. CRISPR/Cas9 system is based on a natural immune system used by the bacteria to protect themselves against viral infection. In the presence of viral DNA within the nucleus, the bacteria produce two types of short RNA, CRISPR RNA (crRNA) that contains the sequence matches the invading viral DNA and transactivating CRISPR RNA (tracrRNA). These two RNAs form a complex with Cas9 nuclease protein that cut DNA. When a matched sequence, known as a guide RNA (gRNA) finds its target within the viral genome, the Cas9 cuts the target DNA, disabling the virus. The use of CRISPR/Cas9 in genome editing of plants is one of the most rapidly emerging technologies in biosciences. More importantly, CRISPR/Cas9 is becoming a user-friendly tool for development of non-transgenic genome edited crops to cope up with changing climate and ensure future food security.
- To develop CRISPR vectors targeting the potential susceptible genes: In silico design of sgRNA for each gene will be done using OMICtools and cocoa genome databases. A comprehensive evaluation on off-target and on-target effects of sgRNA will be done. sgRNA containing target spacer for each gene will be cloned into the vector pRGEB31 as successfully applied for cocoa genome modification.
- To transform cocoa protoplasts with CRISPR vectors: Embryogenic cell suspension (ECS) culture will be developed according to the method described by Shivani et al. (2017). DNA will be extracted from the putative mutant and subjected to PCR with gene-specific primers. The PCR products will be sent for sequencing to confirm the existence of mutations or intact DNA sequences.
- To assess Phytophthora-resistant in cocoa: To evaluate the resistance to Phytophthora, the transgenic seedlings will be used for greenhouse trial according to the method described by Monteiro et al. (2013).
Evidence / Results
CRISPR/Cas9-mediated knockout of targeted genes may lead to Phytophthora infection resistance. Therefore, we hope the simultaneous editing of these potential susceptible genes via CRISPR/Cas9 system enhances Phytophthora-resistant in cocoa crop.
Wael M K Alsultan, Malaysia