Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions   

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Original research article

A brief version of this protocol appeared in:
Plant Methods
Nov 2016


Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include how to design a construct using Golden Gate cloning for targeting two sites, allowing a precise deletion to be introduced into the target gene. The transformation protocol is explained, as are the methods for screening using band shift assay and/or restriction site loss.

Keywords: CRISPR-Cas, Diatom, Thalassiosira pseudonana, Golden Gate, U6 promoter, Band shift assay, Restriction site loss, Phaeodactylum tricornutum


CRISPR-Cas is fast becoming a key method for molecular research. Based on a viral defence mechanism found in bacteria and archaea, CRISPR-Cas induces double-strand breaks (DSBs) at precise locations in the genome. It involves the use of a Cas9 nuclease which forms a complex with a chimeric single guide RNA (sgRNA) formed from CRISPR RNA (crRNA) and trans-activating crRNA (trRNA). Specificity is provided by a 20 nt sequence in the crRNA which corresponds with the target in the genome and guides Cas9 to the correct site by base complementarity. This means that the system is easily programmable and can be applied to new targets simply by changing the 20 nt sequence, provided that a protospacer adjacent motif (PAM) is present in the genome directly following the 20 nt target sequence. For the commonly used Cas9 isolated from Streptococcus pyogenes the PAM sequence is NGG. Gene editing is then achieved either by introducing mutations following imperfect repair by non-homologous end joining (NHEJ), cutting two sites and introducing a precise deletion or through homologous recombination. Since its application in the first eukaryotic systems (Cong et al., 2013; Mali et al., 2013), CRISPR-Cas has been used for genome editing in a wide range of organisms including two diatom species (Hopes et al., 2016; Nymark et al., 2016). Nymark et al. (2016) introduced mutations into the genome of Pheodactylum tricornutum using individual sgRNAs–the protocol for which can be found within Bio-protocol (Nymark et al., 2017). The protocol published in this paper focuses on gene editing in Thalassiosira pseudonana using two sgRNAs to introduce a precise deletion and allow simple screening using the band-shift assay previously described for identifying mutants in higher plants (Brooks et al., 2014). In addition, this method uses Golden Gate cloning (Weber et al., 2011; Engler et al., 2014)–a flexible modular system which allows sequences and cassettes to be easily interchanged and multiple modules to be assembled at once. Whilst this protocol describes targeting of two sites in the same gene to introduce a deletion, the construct can easily be altered to target different genes or greater numbers of genes as previously shown by Sakuma et al. (2014), who demonstrated knock-out of 7 genes using the Golden Gate cloning system.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Hopes, A., Nekrasov, V., Belshaw, N., Grouneva, I., Kamoun, S. and Mock, T. (2017). Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions. Bio-protocol 7(23): e2625. DOI: 10.21769/BioProtoc.2625.

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