Establishment Of Schizochytrium CRISPR/Cas9 System And Its Application In Fatty Acid Metabolism

Docosahexaenoic acid(DHA)is a kind of important n-3 polyunsaturated fatty acids,which has an anti-cancer,anti-inflammatory,prevention of cardiovascular disease effects,and can also promote the development and maturation of infants and young children's nervous system and vision system.And Schizochytrium limacinum is a kind of marine fungus with rich DHA,which belongs to Thraustochytrids family and is now known as its highest DHA content.Due to its important economic value,it has been attracted much attention.Therefore,through the study of Schizochytrium DHA synthesis pathway,to maximize Schizochytrium DHA production by biological engineering method that has very important biological significance and economic value.CRISPR/Cas9 is a new efficient and accurate gene editing technology at present,this technology has been applied successfully in many species gene editing.This paper aims to use Schizochytrium sp.FIO-1 as the research object,by constructing the corresponding editing system of CRISPR/Cas9 gene,knocking out the gene which related to the fatty acid synthesis of Schizochytrium,and this gene will finally be replaced by the Zeocin resistance gene fragment.This will be a preliminary exploration of tje CRISPR/Cas9 gene editing system using in marine fungi Schizochytrium sp.,and the effect on fatty acid production after knocking out the related gene.Accordingly,the main contents of this experiment and research results are as follows:(1)In order to determine the marker gene in this experiment that can successfully screened transformants,some antibiotics which were commonly used in genetic engineering(Zeocin,ampicillin and kanamycin)were selected,to study the influence of the Schizochytrium sp.FIO-1.The Schizochytrium sp.FIO-1 growth results showed that the concentration of 50 ?g/m L and 100 ?g/mL ampicillin and kanamycin had no effect on Schizochytrium's growth,but the growth was inhibited in 50 ?g/mL Zeocin,while the concentration of Zeocin run up to 100 ?g/mL,the growth was inhibited completely and after tenth days,there were still no colonies.As a result,100 ?g/mL Zeocin were selected as the resistance of Schizochytrium screening reagents.(2)According to the design website https://www.dna20.com/products/crispr,a special gRNA sequence were designed applying to the knockout of Schizochytrium PKS subunit PksD,and then constructed this sequence into a efficient expression vector of CRISPR/Cas9 system named pCRISPR-Schi.Then using overlap extension PCR cloned a fragment which contained Zeocin fragment and PksD gene with resistance genes upstream and downstream homologous sequences,then used electroporation method successfully transfer the fragment and vector into Schizochytrium.Through the determination of biomass of transformation strains and wild type strains,there were not much difference in cell dry weight,indicating that the inserted of CRISPR vectors and resistance gene fragment had little effect in the normal growth of the strain,this conclusion also further indicated that CRISPR/Cas9 gene editing system is suitable for the breeding of Schizochytrium limacinum,and can fastly,efficiently knock out or add related genes of Schizochytrium.(3)The fatty acid contents about the successful transformation of Schizochytrium positive strains were measured by gas chromatography-mass spectrometry(GC-MS),results showed that the total lipid content of each strain were improved to a certain extent,the fatty acid composition did not change compared with the wild type strain transformed strains,but the DHA content was reduced about 20%,which indicating that the disruption of PksD gene affected the synthesis of total lipid and DHA,some conclusions were furthermore determined that PksD gene plays an important role in the PKS pathway of Schizochytrium fatty acid synthesis.This system laid the foundation for the further application of CRISPR/Cas9 system study of Schizochytrium gene function.