Torfinn Sparstad

Bio: Torfinn Sparstad is an academic researcher from Norwegian University of Science and Technology. The author has contributed to research in topics: Cas9 & CRISPR. The author has an hindex of 6, co-authored 9 publications receiving 351 citations.

A CRISPR/Cas9 system adapted for gene editing in marine algae

Abstract: Here we report that the CRISPR/Cas9 technology can be used to efficiently generate stable targeted gene mutations in microalgae, using the marine diatom Phaeodactylum tricornutum as a model species. Our vector design opens for rapid and easy adaption of the construct to the target chosen. To screen for CRISPR/Cas9 mutants we employed high resolution melting based PCR assays, mutants were confirmed by sequencing and further validated by functional analyses.

Membrane‐trafficking RabA4c involved in the effect of glycine betaine on recovery from chilling stress in Arabidopsis

Abstract: Glycine betaine (GB) can confer tolerance to several types of stress at low concentrations, either after application to plants or in transgenics engineered to overproduce GB. Based on earlier studies on levels of GB in plants and evidence for effects on gene expression, we hypothesized that at least part of this effect could be ascribed to the activation of the expression of stress tolerance genes. Using a strategy based on high-throughput gene expression analysis with microarrays followed by confirmation with northern blots, we identified Arabidopsis genes upregulated in roots that reinforce intracellular processes protecting cells from oxidative damage and others that appear to be involved in reinforcing a scavenging system for reactive oxygen species (ROS) in cell walls. Upregulated genes in roots include those for the membrane-trafficking RabA4c, the root-specific NADPH-dependent ferric reductase (FRO2) localized to the plasma membrane, mitochondrial catalase 2 and the cell wall peroxidase ATP3a. Comparative studies with wild-type Arabidopsis and knockout mutants for the membrane-trafficking RabA4c gene demonstrated that the mutants respond only slightly to GB, if at all, compared with wild-type in relation to root growth recovery after chilling stress, demonstrating the role of RabA4c in relation to the GB effect. The results point toward links between oxidative stress, gene expression, membrane trafficking and scavenging of ROS such as superoxide and hydrogen peroxide in relation to GB effects on chilling tolerance in plants.

Transgene-free genome editing in marine algae by bacterial conjugation – comparison with biolistic CRISPR/Cas9 transformation

Abstract: The CRISPR/Cas9 technology has opened the possibility for targeted genome editing in various organisms including diatom model organisms. One standard method for delivery of vectors to diatom cells is by biolistic particle bombardment. Recently delivery by conjugation was added to the tool-box. An important difference between these methods is that biolistic transformation results in transgene integration of vector DNA into the algae genome, whereas conjugative transformation allows the vector to be maintained as an episome in the recipient cells. In this study, we have used both transformation methods to deliver the CRISPR/Cas9 system to the marine diatom Phaeodactylum tricornutum aiming to induce mutations in a common target gene. This allowed us to compare the two CRISPR/Cas9 delivery systems with regard to mutation efficiency, and to assess potential problems connected to constitutive expression of Cas9. We found that the percentage of CRISPR-induced targeted biallelic mutations are similar for both methods, but an extended growth period might be needed to induce biallelic mutations when the CRISPR/Cas9 system is episomal. Independent of the CRISPR/Cas9 vector system, constitutive expression of Cas9 can cause re-editing of mutant lines with small indels. Complications associated with the biolistic transformation system like the permanent and random integration of foreign DNA into the host genome and unstable mutant lines caused by constitutive expression of Cas9 can be avoided using the episomal CRISPR/Cas9 system. The episomal vector can be eliminated from the diatom cells by removal of selection pressure, resulting in transient Cas9 expression and non-transgenic mutant lines. Depending on legislation, such lines might be considered as non-GMOs.

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.

Gene regulation of lipid and phospholipid metabolism in Atlantic cod (Gadus morhua) larvae.

Abstract: The mechanism of essentiality of dietary phospholipid (PL) for larval fish is not clear. The main objective of the present study was to determine if the PL requirement of Atlantic cod larvae was due to any genetic impairment caused by functional immaturity. Cod larvae were sampled at 1, 3, 8, 13, 17, 18, 30, 42 and 60 days post hatch (dph) for transcriptome analysis using a recently developed microarray. The fatty acid profile and gene expression levels of cod larvae at 17 dph were compared after feeding differently enriched rotifers, which contained different DHA levels in PL. No significant differences (p<0.05) were found for the two rotifer diets in the overall gene expression level of cod larvae, their growth and survival, and their DHA levels in total lipid and PL fraction. The fatty acid data suggested that dietary EPA was elongated to DPA by cod larvae, and a threshold DHA level in PL to maintain membrane fluidity and other functions may exist. There appeared to be no major effect of development on the expression of key genes of PL biosynthesis suggesting no genetic constrain in early developmental stages. Our overall data suggested that besides the possible limited de novo PC synthesis ability in the intestine, other metabolic constraints should also be considered, especially the possible low input of bile PC as a result of immature liver. Further studies are needed to elucidate the gene expression level and enzyme activity in the PL biosynthesis pathways for specific tissue or cells.

Agricultural uses of plant biostimulants

Abstract: Plant biostimulants are diverse substances and microorganisms used to enhance plant growth. The global market for biostimulants is projected to increase 12 % per year and reach over $2,200 million by 2018. Despite the growing use of biostimulants in agriculture, many in the scientific community consider biostimulants to be lacking peer-reviewed scientific evaluation. This article describes the emerging definitions of biostimulants and reviews the literature on five categories of biostimulants: i. microbial inoculants, ii. humic acids, iii. fulvic acids, iv. protein hydrolysates and amino acids, and v. seaweed extracts. The large number of publications cited for each category of biostimulants demonstrates that there is growing scientific evidence supporting the use of biostimulants as agricultural inputs on diverse plant species. The cited literature also reveals some commonalities in plant responses to different biostimulants, such as increased root growth, enhanced nutrient uptake, and stress tolerance.

Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications.

Abstract: Various compatible solutes enable plants to tolerate abiotic stress, and glycinebetaine (GB) is one of the most-studied among such solutes. Early research on GB focused on the maintenance of cellular osmotic potential in plant cells. Subsequent genetically engineered synthesis of GB-biosynthetic enzymes and studies of transgenic plants demonstrated that accumulation of GB increases tolerance of plants to various abiotic stresses at all stages of their life cycle. Such GB-accumulating plants exhibit various advantageous traits, such as enlarged fruits and flowers and/or increased seed number under non-stress conditions. However, levels of GB in transgenic GB-accumulating plants are relatively low being, generally, in the millimolar range. Nonetheless, these low levels of GB confer considerable tolerance to various stresses, without necessarily contributing significantly to cellular osmotic potential. Moreover, low levels of GB, applied exogenously or generated by transgenes for GB biosynthesis, can induce the expression of certain stress-responsive genes, including those for enzymes that scavenge reactive oxygen species. Thus, transgenic approaches that increase tolerance to abiotic stress have enhanced our understanding of mechanisms that protect plants against such stress.