Developing and Implementing the CRISPR-Cas based gene editing tools in oleaginous microalgae Nannochloropsis oceanica for metabolic engineering.
The exploitation of non-renewable energy sources has impacted the environment and consequent problems are rising in an alarming rate. The concept of biofuels emerged as an eco-friendly and renewable alternative to the crude oil based fuels. Over the years, the research focussed on improving the sustainability of the established biofuel production platform.
The “third generation” biofuel implementing microalgae for production was a clear improvement over its predecessors. However, the “first generation” and “second generation” biofuels account for 99% of the biofuels produced globally.
The prime reason for this is the limited technology available to exploit the complete potential of microalgal strains. Certain oleaginous microalgal strains such as Nannochloropsis oceanica, have the potential to accumulate oils up to 60% of its dry weight under defined conditions.
Nevertheless, the oil accumulation occurs with reduced growth rate which is detrimental from the industrial perspective. Lack of an efficient genome editing tool was the prime reason that hindered the development of industrial relevant microalgal mutants that could uplift the production of most sustainable “third generation” biofuels.
Since its inception in 2013 as a genome editing tool, CRISPR-Cas systems has revolutionized the genetic engineering in wide range of organisms. However, the application of this tool in the microalgal species were stalled by the hypothesised toxic effect of Cas9 in these organisms. In 2016, this dilemma was overcome with limited success when the plasmid based Cas9 expression in the host strains were replaced by transformation of Cas9 protein along with targeting guide RNAs [Ribonucleoprotein; RNP]. Even though the RNP based approach was a milestone in application of CRISPR in microalgae, the unfeasibility in implementing CRISPRi with this strategy was a drawback.
We focus on exploiting the complete potential of existing CRISPR-Cas systems to develop efficient genome editing tools for microalgae N. oceanica. These tools will be later implemented for metabolic engineering of this species for TAG (Triacyl glycerol) and PUFA (Poly unsaturated fatty acids) production.
The student working on this project will have the opportunity to gain hands on experience on following techniques and more:
- Culturing of microalgal strains in lab scale conditions
- Transformation of microalgal strains
- Construction of vectors and cloning in E.coli
- Heterologous protein expression in N. oceanica
- Trials for CRISPR-Cas based genome editing in N. oceanica
- Implementing CRISPR-Cas based tools for metabolic engineering and subsequent characterization of mutants
- Metabolic modelling, a combination of dry lab and wet lab methodology.