CRISPR/Cas9-Mediated Genome Editing via Homologous Recombination in a Centric Diatom Chaetoceros muelleri .

Chaetoceros , the most abundant genus of marine planktonic diatoms, can be used in mariculture. An effective genetic transformation system with a short transformation period was established in Chaetoceros muelleri by electroporation in our previous study. In this study, a sequence-specific clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 vector applicable for C. muelleri was constructed, and the expressions of sgRNA, resistance gene, and Cas9 gene were driven by the endogenous promoters U6 , acetyl-CoA acetyltransferase, and fucoxanthin chlorophyll a/c binding protein , respectively, in the vector. Nitrate reductase ( NR ) and urease ( URE ) genes were edited in C. muelleri , and the NR knockout and NR / URE double-knockout lines displayed the strict auxotrophic phenotype. In addition, the DNA double-strand break was repaired by homologous recombination when a donor DNA was introduced. CRISPR/Cas9 technology was successfully applied to C. muelleri with an editing efficiency of up to 86%, providing a molecular tool for the study of basic biology in C. muelleri and its synthetic biology applications.