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: CRISPR/Cas9 technology, Diatoms, Phaeodactylum tricornutum, Biolistic transformation, HRM analyses
Background
The CRISPR/Cas9 system has proven to be a very efficient and successful genome editing system in a number of eukaryotic organisms, now also including microalgae (Hopes et al., 2016; Nymark et al., 2016; Shin et al., 2016). The CRISPR/Cas9 system includes a guide RNA (gRNA) and a nuclease called Cas9 (Sander and Joung, 2014). These two molecules form a complex where the gRNA directs the complex to the target of interest. The Cas9 nuclease induces double strand brakes at the target site that can be repaired by nonhomologous end joining (NHEJ) which can result in indel mutations, or via the homology-directed repair (HDR) pathway that can be exploited to create defined alterations of the DNA. The presented protocol is the first to describe a step-by-step procedure for applying the CRISPR/Cas9 system to create gene-targeted mutations (indels ranging from one to hundreds of nucleotides) in one of the main diatom model species, P. tricornutum.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
HRM analysis is a post-PCR method that can be performed using the LightCycler® 96 software 1.1 (Roche) enabling easy screening of genetic variations in the PCR amplicons based on differences in the melting points of the amplicons. When performing HRM analysis, at least two technical replicates should be included per sample. Before the HRM analysis, the amplification curves and the melting curves from qPCR raw data should be evaluated. Amplification curves with high crossing point (Cp) values may contain unspecific PCR products, and curves giving > 30 Cp units should be excluded. The cycling conditions need to be optimized if the melting curves indicate unspecific PCR products in the WT samples or in the Non-template controls (NTCs). The crossing point between samples should not vary more than 5 Cp units. Mutations indicated by the HRM analyses must be confirmed by sequencing.
Recipes
Acknowledgments
This work was funded by a grant from the Gordon and Betty Moore Foundation GBMF 4966 to Atle Bones and the NTNU enabling technologies program.
References
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