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.

Other RNA guided nucleases have been tested by electroporation with in vitro produced nuclease with sgRNA complexes. Non-cutting dCas9 is being tested as a platform for other genetic engineering techniques.

Inducible expression is possible using the nitrite reductase promoter or the bidirectional nitrate reductase/nitrate transporter promoter.

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

Multiple marker-free methods have been demonstrated in Nannochloropsis. The first is by expression of the CRISPR components from a non-integrating episomal plasmid. The episome is maintained by Nannochloropsis when antibiotic selection is applied, but the episome is lost when selection is removed. Transformants can therefore be screened for the Cas9 protein, then examined for a mutation, and lines with mutations are removed from antibiotic selection to cure them of the CRISPR episome

A second marker-free method by marker recycling was developed by Synthetic Genomics Inc, wherein an antibiotic marker is inserted in the genomic target site following cutting by CRISPR. The insertion contains Cre-Lox sites and inducible expression of the Cre recombinase excises the transgenic sequence.

UCan CRISPR module

Choosing a guide and designing sgRNA

Sequences of ~20 bp upstream of a PAM sequence (NGG) are identified in the 5' end of the target gene or functional domain. The identified sequences (target + PAM) are submitted to the CCMP1779 BLAST search and those with additional matches on the 3' end are discarded. The Nannochloropsis oceanica IMET1 genome is available in Chop Chop for automated identification of target sites. Selection of sgRNAs for N. oceanica CCMP1779 can be automatically conducted using the http://crispor.tefor.net/ web tool.

Obtaining a CRISPR vector

A vector for CRISPR based gene disruption is available from Addgene. This vector provides ampicillin resistant in E. coli and hygromycin resistance in Nannochloropsis. It is maintained as an episome in Nannochloropsis when transformed as a circular plasmid.

Options for cloning of the ribozyme-sgRNA in the TypeIIS cloning site v1.0 of the pNoc ARS CRISPR vector series

For the incorporeation of the customized hammerhead sequence and the gRNA into the cloning site, the cloning of a ca. 65 bp long fragment is required. Several options may be used for this:

Two-step cloning

(i) Site directed mutagenesis/ Two-step cloning approach for the subcloning of the hammerhead and guide sequence as described by Poliner et al. (2018),

BspQI ligation

(ii) the design of a complementary primer pair with overhanging nucleotides for cloning via the BspQI restriction sites and

Gibson Assembly

(iii) PCR amplification of a specific product using 20-25bp bases binding to the unmodified part of the hammerhead sequence and target specific overhangs (first 6bp of the hammerhead sequence and sgRNA) and cloning via Gibson Assembly or BspQI restriction sites.

Transformation

Screening

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