Cas9-guide RNA ribonucleoprotein-induced genome editing in the industrial green alga Coccomyxa sp. strain KJ

Abstract

BackgroundOxygen-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.ResultsIn 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%.ConclusionsOur study shows applicability of DNA-free genome editing in Coccomyxa, which may be applicable in other Trebouxiophyceae species.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1327-1) contains supplementary material, which is available to authorized users.

Efficient and multiplexable genome editing using Platinum TALENs in oleaginous microalga, Nannochloropsis oceanica NIES‐2145

“…Recently, CRISPR‐Cas9 ribonucleoprotein (RNP) systems have been applied in some microalgae (Greiner et al, 2017; Naduthodi et al, 2019; Nomura et al, 2019; Yoshimitsu et al, 2018). The introduction of CRISPR‐Cas9 RNP has the advantage of avoiding plasmid insertion.…”

“…This barrier is likely to be overcome by breeding of high‐performance alga that exhibits fast growth and high‐lipid productivity. To this end, various studies of algal molecular breeding for the efficient biodiesel production are actively proceeding (Chen et al, 2019; Gomaa, Al‐Haj, & Abed, 2016; Takahashi et al, 2018; Yoshimitsu, Abe, & Harayama, 2018).…”

Development of a species-specific transformation system using the novel endogenous promoter calreticulin from oleaginous microalgae Ettlia sp.

“…The construction of a plasmid for the stable expression of gRNA and Cas9 requires an endogenous promoter and codon optimization of the Cas9 protein. In this case, pEtt-CRT and pEtt-psaD could easily be applied to construct plasmids 38 – 40 . A ribonucleoprotein (RNP) is currently preferred over a plasmid for genome editing due to its low toxicity, low off-target effects, and easy preparation without plasmid construction.…”

Genetic Engineering Strategies for Euglena gracilis and Its Industrial Contribution to Sustainable Development Goals: A Review

“…Strains created using the genome editing technology employing the ribonucleoprotein (RNP) complex are transgenefree and are suitable for industrial use. At present, RNP-based genome editing of microalgae using CRISPR-Cas9 (Cong and Zhang, 2014) has been actively performed in various species, but many selection-free methods display a low mutation efficiency (∼1%) (Table 2; Baek et al, 2016;Shin et al, 2016;Ferenczi et al, 2017;Serif et al, 2018;Yoshimitsu et al, 2018;Findinier et al, 2019;Naduthodi et al, 2019;Nomura et al, 2019). In E. gracilis, a highly efficient genome editing method was recently established based on a Cas9-RNP introduction procedure, which does not depend on the expression of the transgene in the cell (Figure 2).…”