CRISPR/Cas9-induced ?-carotene hydroxylase mutation in Dunaliella salina CCAP19/18

Abstract Dunaliella salina ( D. ) has been exploited as a novel expression system for the field of genetic engineering. However, owing to low or inconsistent target proteins, it greatly restricted practical production recombinant proteins. Since accurate gene editing function clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system, ?-carotene hydroxylase was chosen an example explore application with purpose improving level foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed targeting exon 1 and 3 CCAP19/18 (Dschyb). mutants obtained by salt gradient transformation method, Dschyb identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content analyzed high-performance liquid chromatography at different time points after high intensity treatment. Compared wild type strains, levels showed significant increase, nearly up 1.4 ?g/ml, zeaxanthin decreased various degrees in mutants. All results provide compelling evidence targeted . This study gave first successful which very important significance increasing carotene yield meeting realistic industry demand. Furthermore, provides approach overcome current obstacles , then gives strong tool facilitates development system.