CRISPRi mediated phosphoenolpyruvate carboxylase regulation to enhance the production of lipid in Chlamydomonas reinhardtii

In this study, CRISPRi (clustered regularly interspaced short palindromic repeats interference) was used for the first time to regulate expression of exogenously supplied rfp gene as a proof-of-concept, and endogenous PEPC1 gene as a proof-of-function in Chlamydomonas reinhardtii. The efficiency of 94% and stability of 7 generations via CRISPRi mediated gene regulation in C. reinhardtii have been demonstrated by RFP. Gene PEPC1 encoding proteins are essential for controlling the carbon flux that enters the TCA cycle and plays a crucial role in carbon partitioning of substrates in competition with lipid synthesis. All CrPEPC1 down-regulated strains have lower chlorophyll color, but higher biomass concentration and lipid accumulation rate. The present results revealed that CRISPRi based transcriptional silencing was applicable in C. reinhardtii and expanded the way to improve the yield, titer and productivity of microalgae-based products.