Cas9 RNP mediated HDR for nitrate reductase

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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.

• Naduthodi MIS, Mohanraju P, Südfeld C, D'Adamo S, Barbosa MJ, van der Oost J. CRISPR-Cas ribonucleoprotein mediated homology-directed repair for efficient targeted genome editing in microalgae. Biotechnology for biofuels. 2019; 12:66.