Research
| Title： | CRISPR/Cas9-Mediated Genome Editing via Homologous Recombination in a Centric Diatom Chaetoceros muelleri |
|---|---|
| First author： | Yin, Wenxiu; Hu, Hanhua |
| Journal： | ACS SYNTHETIC BIOLOGY |
| Years： | 2023 |
| DOI： | 10.1021/acssynbio.3c00051 |
| Abstract： | 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. |
