Abstract
To date, Chlorella vulgaris is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of C. vulgaris UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (NR) and adenine phosphoribosyltransferase (APT). Genome-edited mutants, nr and apt, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in C. vulgaris UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants, nr can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of C. vulgaris.
Highlights
Accepted: 3 January 2021Microalgae are photoautotrophic eukaryotic organisms that have potential as producers of biofuel and biomaterials, and significantly contribute to ecological processes by reducing atmospheric CO2 concentrations [1,2,3]
adenine phosphoribosyltransferase (APT), coding for a protein that catalyzes the conversion of adenine to adenylate, was chosen as a target for genome editing because it is recognized as a promising marker gene
Targeting these genes in C. vulgaris UTEX395 facilitates the selection of mutant strains, as the disruption of nitrate reductase (NR) or APT results in uninhibited growth in medium containing KClO3 or 2-fluoroadenine (2-FA), respectively [29,37]
Summary
Microalgae are photoautotrophic eukaryotic organisms that have potential as producers of biofuel and biomaterials, and significantly contribute to ecological processes by reducing atmospheric CO2 concentrations [1,2,3]. Among microalgal species, those belonging to the Chlorella genus, the most widely used microalgae in industry, are considered promising hosts for the expression of therapeutic proteins or the production of high-value compounds [4]. Despite the advantages of Chlorella spp., the instability of protein expression and the relatively low production yield of high-value chemicals impede the expansion of its commercial use [4,6]. Electroporation and bombardment are mainly used to transfer genes into cells, and antibiotics such as hygromycin and zeocin are used for the selection of transformants [7,8,9,10,11,12,13,14,15,16]
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