Establishing And Optimizing The CRISPR/Cas9 Gene-editing Process In Chlorella

Chlorella is a unicellular eukaryotic green alga with efficient photosynthesis.It can be used as a bioreactor to synthesize complex proteins and to generate active molecules.Chlorella has many features,such as rapid growth,rapid reproduction rate,simple and cost-effective artificial culture condition,and easy-to-large-scale culture.Chlorella is also known as green human nutrition source for the 21 st century,because it contains high level of lipids,vitamins,amino acids,minerals and proteins,and,it is also a very good source of single cell protein(SCP).Given the above advantages,chlorella has a wide range of applications such as treatment of sewage,biodiesel,feed and food,medicinal value and so on.CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas(CRISPR-associated)is an RNA-directed Cas nuclease system to target gene and make specific DNA modification.The type II CRISPR/Cas immune system relies on the Cas9 endonuclease family for targeting and cleaving exogenous DNA.CRISPR/Cas9 system has become a research hotspot given its advantages such as relatively simple construction,low cost,high precision,and the ability to edit several gene loci simultaneously.In this study,the growth characteristics of Chlorella Vulgaris were investigated by measuring growth curve,Specific Growth Rate(SGR),and density curve.And the genetic engineering screening marker of Chlorella vulgaris and Gene transformation system were optimized.The plant binary expression vector pCAMBIA1302 that contains Hygromycin resistant gene was selected as the main vector in this study,and the gRNA targeting the Hygromycin gene of pCAMBIA1302 was designed.The recombinant plasmid p RGE31-gRNA was successfully constructed by inserting the gRNA sequence into plasmid pRGE31,a plasmid containing spCas9.Plasmids pCAMBIA 1302 and pRGE31-gRNA were co-transformed into Chlorella vulgaris via electroporation,and transient expression was detected,indicating the successful establishment of the CRISPR/Cas9 gene transformation system in Chlorella.This study successfully established the exogenous gene editing system of Chlorella,and used the CRISPR / Cas9 system to edit Hygromycin gene in Chlorella.The protocol established in this study provides the experimental basis for the development of endogenous gene editing as well as molecular-based breeding techniques in Chlorella.