CRISPR/Cas9 Gene Editing in the Marine Diatom Phaeodactylum tricornutum

Abstract

The establishment of the CRISPR/Cas9 technology in diatoms ( Hopes et al., 2016 ; Nymark et al., 2016 ) enables a simple, inexpensive and effective way of introducing targeted alterations in the genomic DNA of this highly important group of eukaryotic phytoplankton. Diatoms are of interest as model microorganisms in a variety of areas ranging from oceanography to materials science, in nano- and environmental biotechnology, and are presently being investigated as a source of renewable carbon-neutral fuel and chemicals. Here we present a detailed protocol of how to perform CRISPR/Cas9 gene editing of the marine diatom Phaeodactylum tricornutum, including: 1) insertion of guide RNA target site in the diatom optimized CRISPR/Cas9 vector (pKS diaCas9-sgRNA), 2) biolistic transformation for introduction of the pKS diaCas9-sgRNA plasmid to P. tricornutum cells and 3) a high resolution melting based PCR assay to screen for CRISPR/Cas9 induced mutations.

Keywords: Biolistic transformation; CRISPR/Cas9 technology; Diatoms; HRM analyses; Phaeodactylum tricornutum.

Figure 1.. Example of adapter with 5’ TCGA and AAAC overhangs

Figure 2.. Schematic illustration of the pKS diaCas9-sgRNA plasmid.

A diatom codon optimized Cas9 is placed under control of a promoter for a fucoxanthin chlorophyll a/c binding gene (LHCF2), and the expression of the sgRNA is driven by the P. tricornutum U6 promoter. Two BsaI cutting sites are located within the sgRNA cassette to enable insertion of a small adapter for targeting the gene of interest ( Nymark et al., 2016 ).

Figure 3.. Overview of the screening procedure for detecting CRISPR/Cas9 induced mutations.

A. Wait 2-4 weeks after biolistic transformation of P. tricornutum cells with the pKS diaCas9-sgRNA plasmid for colonies to appear on selection plates; B. Transfer the desired number of putative mutant colonies to a 24 or 48-well plate. Grow the cells for 1-2 weeks. C. Remove a small amount of cells from each well and lysate the cells; D. Use the lysate as template and amplify a 500-1,000 bp region around the target site by PCR; E. Check the PCR products by gel electrophoresis to identify large indels; F. Dilute the PCR product 1:4 x 10⁶; G. Perform HRM analyses to detect smaller indels. Use the diluted PCR product from (F) as template in a PCR reaction that amplifies a region of around 100 bp surrounding the target site. H. Analyze the HRM data. Identify samples with CRISPR/Cas9 induced mutations by studying the difference in melting temperature between mutants and WT samples. I. Confirm mutations detected by HRM analyses by sequencing.

Figure 4.. HRM data presented as normalized melting peaks.

Purple line: profile of a WT PCR product. Red line: profile of a PCR product containing a single biallelic G-insert. Yellow line: profile of a PCR product containing a single biallelic A-insert. Brown line: profile of a mix of PCR products containing a variety of deletions of different sizes.