Genome editing with removable TALEN vectors harboring a yeast centromere and\xa0autonomous replication sequence in\xa0oleaginous microalga

Tomokazu Kurita,Akihide Takami,Tetsushi Sakuma,Shinichiro Maeda,Masako Iwai,Fumihiko Saito,Seiji Nomura,Takashi Yamamoto,Atsushi Sakamoto,Kumiko Okazaki,Keishi Moroi,Hiroyuki Ohta

doi:10.1038/s41598-022-06495-y

Abstract

Algal lipids are expected to become a basis for sustainable fuels because of the highly efficient lipid production by photosynthesis accompanied by carbon dioxide assimilation. Molecular breeding of microalgae has been studied to improve algal lipid production, but the resultant gene-modified algae containing transgenes are rarely used for outdoor culture because the use of genetically modified organisms (GMOs) is strictly restricted under biocontainment regulations. Recently, it was reported that plasmids containing yeast centromere and autonomous replication sequence (CEN/ARS) behaved as episomes in Nannochloropsis species. We previously reported that the Platinum TALEN (PtTALEN) system exhibited high activity in Nannochloropsis oceanica. Therefore, we attempted to develop a genome editing system in which the expression vectors for PtTALEN can be removed from host cells after introduction of mutations. Using all-in-one PtTALEN plasmids containing CEN/ARS, targeted mutations and removal of all-in-one vectors were observed in N. oceanica, suggesting that our all-in-one PtTALEN vectors enable the construction of mutated N. oceanica without any transgenes. This system will be a feasible method for constructing non-GMO high-performance algae.

Highlights

The development of sustainable energy production and reduction of carbon dioxide emissions are vital to protect the environment
The genome editing activity of the all-in-one Platinum TALEN (PtTALEN)-ARS plasmid was assayed using the heteroduplex mobility assay (HMA) and Cel-I assay of the PCR products
PCR products derived from all-in-one PtTALEN-ARS plasmid exhibited a band shift in the HMA that was similar to the positive control, suggesting the introduction of mutations (Fig. 1C)

Summary

Introduction

The development of sustainable energy production and reduction of carbon dioxide emissions are vital to protect the environment. Gene manipulation using episomal plasmids is effective because plasmids containing expression cassettes of genome editing tools can be removed from the host cells. Poliner et al reported that CRISPRCas[9] all-in-one vectors containing a CEN/ARS could be removed from Nannochloropsis species host cells by culturing in F2N medium without antibiotics[9]. This strategy is feasible for constructing plasmid-removed genome edited strains. All-in-one PtTALEN plasmids may be inserted into the endogenous genome of the host N. oceanica cells This is inconvenient for outdoor culture and serial gene manipulation, since transformant cells have transgenes containing antibiotic marker genes. The locus effect of plasmid insertion could negatively affect host cell integrity and PtTALEN expression

Methods

Results

Conclusion