CRISPR-Cas ribonucleoprotein mediated homology-directed repair for efficient targeted genome editing in microalgae Nannochloropsis oceanica IMET1

Abstract

Background: Microalgae are considered as a sustainable feedstock for the production of biofuels and other value-added compounds. In particular, Nannochloropsis spp. stand out from other microalgal species due to their capabilities to accumulate both triacylglycerol (TAG) and polyunsaturated fatty acids (PUFAs). However, the commercialization of microalgae-derived products is primarily hindered by the high production costs compared to less sustainable alternatives. Efficient genome editing techniques leading to effective metabolic engineering could result in strains with enhanced productivities of interesting metabolites and thereby reduce the production costs. Competent CRISPR-based genome editing techniques have been reported in several microalgal species, and only very recently in Nannochloropsis spp. (2017). All the reported CRISPR-Cas-based systems in Nannochloropsis spp. rely on plasmid-borne constitutive expression of Cas9 and a specific guide, combined with repair of double-stranded breaks (DSB) by non-homologous end joining (NHEJ) for the target gene knockout.

Results: In this study, we report for the first time an alternative approach for CRISPR-Cas-mediated genome editing in Nannochloropsis sp.; the Cas ribonucleoproteins (RNP) and an editing template were directly delivered into microalgal cells via electroporation, making Cas expression dispensable and homology-directed repair (HDR) possible with high efficiency. Apart from widely used SpCas9, Cas12a variants from three different bacterium were used for this approach. We observed that FnCas12a from Francisella novicida generated HDR-based targeted mutants with highest efficiency (up to 93% mutants among transformants) while AsCas12a from Acidaminococcus sp. resulted in the lowest efficiency. We initially show that the native homologous recombination (HR) system in N. oceanica IMET1 is not efficient for easy isolation of targeted mutants by HR. Cas9/sgRNA RNP delivery greatly enhanced HR at the target site, generating around 70% of positive mutant lines.

Conclusion: We show that the delivery of Cas RNP by electroporation can be an alternative approach to the presently reported plasmid-based Cas9 method for generating mutants of N. oceanica. The co-delivery of Cas-RNPs along with a dsDNA repair template efficiently enhanced HR at the target site, resulting in a remarkable higher percentage of positive mutant lines. Therefore, this approach can be used for efficient generation of targeted mutants in Nannochloropsis sp. In addition, we here report the activity of several Cas12a homologs in N. oceanica IMET1, identifying FnCas12a as the best performer for high efficiency targeted genome editing.

Keywords: CRISPR; Cas12a; Cas9; Genome editing; Homologous recombination; Homology-directed repair; Microalgae; Nannochloropsis; Ribonucleoproteins.

Fig. 1

Approach; a the purified Cas protein is assembled with guide RNA to form the active RNP complex which is transformed along with the editing template into the competent Nannochloropsis oceanica IMET1 cells. The transformed cells are selected on ASW-NB plated with zeocin and ammonia and the mutants are screened by cPCR. b The Cas RNP introduces DSB at the target site which is repaired by HDR in presence of the editing template resulting in targeted mutants

Fig. 2

a Percent of mutants obtained with various RNP-based HDR. b Gel electrophoresis image of the WT, NR-KO and mixed colonies upon colony PCR. c Phenotypic characterization of NR mutants, the NR mutants were observed to bleach in media with nitrate as the sole nitrogen source while they grew similar to WT in media with ammonia as the nitrogen source (The re-streak plates for characterization included in Additional file 1: Data S13). d Sequencing result of a NR-KO mutant showing precise integration of the Zeocin resistance cassette into the host genome knocking out the NR gene