Background: Oxygen-evolving photosynthetic microorganisms, collectively termed as microalgae, are gaining attention as alternative fuel sources. The unicellular alga Coccomyxa sp. strain KJ that belongs to the class Trebouxiophyceae can grow rapidly in minimal mineral media and accumulate triacylglycerols at levels > 60% (w/w) of its dry weight under nitrogen depletion conditions. Thus, the strain can be a good candidate for biofuel production. Still, substantial improvements in lipid productivity and other traits of this strain are needed to meet commercial production requirements. Consequently, the development of new genetic tools including genome editing that are applicable to this strain is highly desired.
Results: In this paper, we report successful genome editing of strain KJ by intracellular delivery of a ribonucleoprotein complex comprising recombinant Cas9 protein and guide RNA. For introduction of Cas9-guide RNA ribonucleoprotein into strain KJ cells, we used an electroporator with a short (2.5 ms) electric pulse at a high field strength (7500 V cm-1) followed by multiple 50-ms electric pulses at low field strength (250 V cm-1). Under these conditions, we successfully isolated several knockout lines of the FTSY gene of strain KJ, encoding a signal recognition particle-docking protein at a frequency of 0.01%.
Conclusions: Our study shows applicability of DNA-free genome editing in Coccomyxa, which may be applicable in other Trebouxiophyceae species.
Keywords: Cas9-gRNA RNP; Coccomyxa; Electroporation; Genome editing; Microalgae.