CRISPR in Nannochloropsis

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CRISPR/Cas9 techniques in Nannochloropsis

Production and screening of Cas9

Cas9 is usually expressed from an endogenous promoter (VCP, Ribi) at moderate levels. SV40 NLS effectively direct the protein to the nucleus. Fusion with fluorescent or luciferase reporters enhances screening for positive transformants.

Cas9 variants

Inducible expression

Production and screening of sgRNA

An effective sgRNA should not have terminal modifications and needs to present in the nucleus. In Nannochloropsis sgRNAs have been produced using protein coding promoters and exogenously produced and transformed into the cells. To generate more efficient sgRNAs from RNA polymerase II promoters, self-cleaving ribozymes can be appended to the sgRNA gene.

Dual sgRNA expression

Screening for mutations

Selection markers, reporter proteins, and sequencing techniques are used to determine if a mutation may be present. Reporter proteins, and Cas9 reporter fusions (GFP, or NanoLuciferase) are useful as proxies for transgenic expression of the other components. Single guide RNAs can be designed to target restriction sites, enabling a high-throughput screening method. Sanger sequencing is needed to confirm mutations are introduced and stable.

Marker-free methods

Published studies

Ajjawi, I., Verruto, J., Aqui, M., Soriaga, L. B., Coppersmith, J., Kwok, K., … Moellering, E. R. (2017). Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator. Nature Biotechnology, 35(7), 647–652. https://doi.org/10.1038/nbt.3865

Poliner, E., Takeuchi, T., Du, Z.-Y., Benning, C., & Farré, E. M. (2018). Nontransgenic Marker-Free Gene Disruption by an Episomal CRISPR System in the Oleaginous Microalga, Nannochloropsis oceanica CCMP1779. ACS Synthetic Biology, 7(4), 962–968. https://doi.org/10.1021/acssynbio.7b00362

Verruto, J., Francis, K., Wang, Y., Low, M. C., Greiner, J., Tacke, S., … Moellering, E. R. (2018). Unrestrained markerless trait stacking in Nannochloropsis gaditana through combined genome editing and marker recycling technologies. Proceedings of the National Academy of Sciences, 201718193. https://doi.org/10.1073/pnas.1718193115

Wang, Q., Lu, Y., Xin, Y., Wei, L., Huang, S., & Xu, J. (2016). Genome editing of model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9. The Plant Journal, 88(6), 1071–1081. https://doi.org/10.1111/tpj.13307

Future directions