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

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.

WITHDRAWN: Environmentally-informed functional characterization of a plastid diatom metabolic bridge of mitochondrial origin

“…S4, S7 ). Establishing the relative fluxes that occur in each direction of cpEnolase and cpPGAM enzymes will depend on the construction of multigene knockouts for different P. tricornutum Enolase and PGAM isoforms (Yin and Hu 2023).…”

Complementary environmental analysis and functional characterization of a plastid diatom lower glycolytic-gluconeogenesis pathway

“…The direction (import or export) and significance of these amino acid fluxes will be best determined e.g. with an inducible knockout mutant compromised for both plastidial glycolysis and amino acid incorporation (e.g., via nitrate reductase) to allow direct metabolic quantification of de novo synthesised amino acids (McCarthy, Smith et al 2017, Yin and Hu 2023).…”

Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-Associated Protein and Its Utility All at Sea: Status, Challenges, and Prospects

“…The knockout of the nitrate reductase gene ( NR ) was performed in T. pseudonana using CRISPR/Cas, and the mutants showed suppressed growth on nitrate [ 162 ]. Recently, CRISPR/Cas9 technology was successfully applied to the nutrient regulation study in the centric diatom Chaetoceros muelleri [ 163 ]. The knockout of NR and urease genes generated single- and double-knockout lines in C. muelleri , which led to an auxotrophic phenotype under different N nutrient conditions, providing a useful tool for future studies on C. muelleri [ 163 ].…”

“…Recently, CRISPR/Cas9 technology was successfully applied to the nutrient regulation study in the centric diatom Chaetoceros muelleri [ 163 ]. The knockout of NR and urease genes generated single- and double-knockout lines in C. muelleri , which led to an auxotrophic phenotype under different N nutrient conditions, providing a useful tool for future studies on C. muelleri [ 163 ]. In green alga, CRISPR/Cas9 technology was also used to construct auxotrophic strains of C. reinhardtii [ 164 ].…”