Development Of CRISPR/Cas9 Genome Editing Techniques In Green Seaweed Ulva Prolifera

CRISPR/Cas9 is a new generation of gene editing technology,which has been rapidly applied in the fields of biological research and genetic breeding.Macroalgae are important objects of marine aquaculture in China,which have outstanding research value and economic value,but gene editing technology of macroalgae has not been established yet.Ulva prolifera is a green seaweed,whose life cycle is short and easy to regulate.Meanwhile,it has various reproductive strategies and can be effectively proliferated in enclosed water.Especially,both of nuclear and chloroplast transformation systems of U.prolifera have been established.Thus,U.prolifera is an ideal material for the development of macroalgae gene editing technology.In this paper,we carried out the research in three aspects of target-gene cloning,gene editing,and verification methods of gene editing with a material of U.prolifera,and we established CRISPR/Cas9 gene editing technology systematically with research progress as follows:Firstly,nitrate reductase gene of U.prolifera(UpNR)was cloned and characterized.According to the U.linza transcriptome data,we successfully cloned the UpNR gene by rapid amplification of cDNA ends and genome-walking.The UpNR was consisted of six introns and seven exons encoding 863 amino acids,which harbors all five essential domains and 21 key invariant residues in NRs.The function of UpNR was verified by qPCR,and the expression characteristics of UpNR under various nitrogen source conditions were also clarified.With these results,we characterized UpNR,and an ideal target point was provided for the establishment of CRISPR/Cas9 gene editing technology in U.prolifera.After that,gene editing of UpNR by CRISPR/Cas9 was achieved in U.prolifera.Firstly,we designed a series of sgRNAs for the UpNR gene,and the activities of mediating gene targeting of sgRNAs were detected and compared in vitro.Secondly,gene editing vectors harboring each sgRNA of high activity were constructed and transformed into U.prolifera,and we had successfully detected the transient transcription of cas9 and sgRNA in vivo.Then,resistant mutants were obtained under the screening of chlorate and basta.At last,with various molecular methods such as the PCR-RE assay,Sanger sequencing,and next-generation sequencing(NGS),we successfully detected six UpNR mutational genotypes caused by indels.The implementation of UpNR gene editing marks the establishment of gene editing technology in U.prolifera for the first time.In the end,we successfully verified the carotenoid synthesis pathway of U.prolifera.The phytoene synthase gene(UpPSY),phytoene desaturase gene(UpPDS),zeta-carotene desaturase gene(UpZDS),and lycopene beta-cyclase gene(UpLCYb)of U.prolifera were successfully cloned and their structures were analyzed.Gene functions of the four genes were verified by genetic complementation experiments.Real-time quantitative PCR results showed that transcript levels of UpPSY,UpPDS and UpZDS were induced by illumination,while changes in nutrient conditions made no significant variation.In contrast,UpLCYb gene was not significantly induced by illumination,but there was a strong expression of this gene in depleted nutrient condition.Thus,we have preliminarily characterized the carotenoid synthetic pathway of U.prolifera,and it laid the foundation for subsequent application of gene editing.In this paper,UpNR,which was the first cloned nitrogen metabolism gene of green macroalgae,was characterized,and gene editing was achieved by using CRISPR/Cas9 in macroalgae for the first time with UpNR gene as a target gene.Meanwhile,we have verified the carotenoid synthesis pathway of U.prolifera,which would be an important target site for the technical evaluation of gene knockout,single base editing,and transcriptional manipulating mediated by CRISPR/Cas9.