Development of CRISPR/Cas9 system in Chlorella species to enhance lipid accumulation

Abstract

Being the green gold of the future microalgae have recently attracted considerable interest worldwide for they can be used to produce different kinds of metabolites such as lipids protein pigments and bioactive compounds In the last decade the efforts attended to enhance high-value compounds in microalgae are motivated by genetic manipulation Among them the CRISPR/Cas9 system appears to be the most efficient and novel technology Compared to ZFN and TALEN this newly developed gene editing tool was easier to work with more affordable and was able to regulate multiple genes simultaneously In this study we aimed to enhance the lipid accumulation in three different species of Chlorella by CRISPR/Cas9-based gene editing tools and other characterization analyses such as growth rate measurement and protein production had been carried out as well In the first part of this study we attempted to determine plasmid with suitable genomic components which could be utilized in the transformation of Chlorella via electroporation We had tested several plasmids which could be used in microalgae Chlamydomonas with hsp70A/rbcS and CaMV35S promoter respectively However these plasmids were failed to transform into Chlorella Finally plasmid pCAMBIA1302 containing right border (RB) left border (LB) from Agrobacterium tumefaciens and a fragment of mGFP was successfully transformed into microalgae Chlorella vulgaris and Chlorella sorokiniana by electroporation under 360 V at 25 ?F and 200 ohm Selected colonies were tested by spectrophotometer and inverted fluorescence microscopy (IFM) with detection at excitation 488 nm and emission 510 nm Strain 29 and 48 from C vulgaris as well as strain 2 4 and 11 from C sorokiniana showed higher fluorescent value compared with wild type (28% and 67% in C vulgaris 46% 58% and 62% in C sorokiniana respectively) proved plasmid with RB/LB is suitable for gene insertion in Chlorella Aside from that pHSE401 containing fragment of Cas9 RB/LB was used as vector with sgRNA designed from fad3 gene which can affect the lipid accumulation in C vulgaris The lipid content of transgenic strains was improved up to 40% On the other hand Chlorella variabilis NC64A had been completely sequenced and certain gene like PEPC has been located in its genomic DNA However its low recovery rate hampered the transformation efficacy and we were not able to proceed its genome editing Due to the lack of genomic information in C sorokiniana and consider most microalgae are in high guanine contents we establish a new approach via high guanine in sgRNA which name “Adaptive Single Guide Assisted Regulation DNA (ASGARD)” Coupled with the CRISPRi and CRISPRa system characterization analyses were conducted to understand how it affect the biomass lipid accumulation and protein productivity Although there was no significant difference between wild type and transformants the protein content of all the transgenic strains was improved up to 60% g/g DCW This novel idea and technology are high potential for gene regulating in microalgae

Date of Award	2019
Original language	English
Supervisor	I-Son Ng (Supervisor)