High-efficiency transformation of a centric diatom Chaetoceros muelleri by electroporation with a variety of selectable markers

Yin, Wenxiu; Hu, Hanhua

doi:10.1016/j.algal.2021.102274

Cited by 12 publications

(10 citation statements)

References 32 publications

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“…HDR efficiency was impacted by sgRNA and donor DNA, and sgRNA with high editing efficiency resulted in high HDR efficiency (Figure S4), while circular donor DNA was more likely to achieve HDR-mediated mutagenesis than linearized plasmids (Figure ). Linearized plasmids could be integrated into the genome via NHEJ more easily than circular ones, and the transformation efficiency was 3- to 7-fold higher in linearized ones by electroporation . It is implied that the plasmid that is not integrated may provide an HR template for the repair of DSBs.…”

Section: Resultsmentioning

confidence: 99%

“…Vectors were introduced to the cells by electroporation as previously described . Linearization of CRISPR/Cas9 vectors, NR overexpression plasmids, and HR plasmids was performed with Kpn I, Nde I, and Nde I, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Lhcf4p was amplified by the primer pair pks-Cas9-Lhcf4p-HR2_fw and pks-Cas9-Lhcf4p-HR2_rv with plasmid pCmACATp-ble as the template. The ble expression cassette (containing ACATp , ble , and FCP A terminator of P.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, the genome sequences of C. muelleri have been released, , and its genetic transformation system has been established by electroporation with high transformation efficiency and a short transformation period (transformants obtained after 4–7 d) in our lab . These studies have illustrated the potential of C.…”

Section: Introductionmentioning

confidence: 98%

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

Yin

2023

ACS Synth. Biol.

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

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

Yin

2023

ACS Synth. Biol.

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“…Additionally, some Chaetoceros species have been used as biological indicators for studying marine environmental change (Wang et al, 2010). Furthermore, many Chaetoceros species have been applied to remove certain antibiotics from wastewater (Mojiri et al, 2021), and C. muelleri has been exploited as a renewable precursor to liquid fuels or as a lipid source because of its high growth rate, tolerance to a broad range of temperatures, and specific conductance and large quantity of intracellular lipid (McGinnis et al, 1997;López-Elías et al, 2005;Yin and Hu, 2021).…”

Section: Introductionmentioning

confidence: 99%