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

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

Automated summary

This study successfully performed targeted gene editing on D. saline using CRISPR/Cas system, significantly increasing the levels of beta-carotene in mutants. These results have important practical implications for increasing carotene yield and meeting industry demand.