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.

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.

Adaptation is the central feature and leading explanation for the evolutionary diversification of life. Adaptation is also notoriously difficult to study in nature, owing to its complexity and logistically prohibitive timescale. We leverage extensive contemporary and historical collections of Ambrosia artemisiifolia—an aggressively invasive weed and primary cause of pollen-induced hayfever—to track the phenotypic and genetic causes of adaptation across its native and invasive ranges in North America and Europe, respectively. Large haploblocks—indicative of chromosomal inversions—contain a disproportionate share of genomic regions conferring parallel adaptation between ranges (18%), are associated with rapidly adapting traits, and exhibit dramatic frequency shifts over space and time. These results highlight the importance of structural and large-effect variants in rapid adaptation, which have been critical to A. artemisiifolia’s global spread.One Sentence SummaryParallel evolution between native and invasive ranges of Ambrosia artemisiifolia is aided by putative chromosomal inversions.

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 nanoand 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.

Summary Phosphorus (P) is one of the limiting macronutrients for algal growth in marine environments. Microalgae have developed adaptation mechanisms to P limitation that involve remodelling of internal phosphate resources and accumulation of lipids. Here, we used in silico analyses to identify the P‐stress regulator PtPSR (Phaeodactylum tricornutum phosphorus starvation response) in the diatom P. tricornutum. ptpsr mutant lines were generated using gene editing and characterised by various molecular, genetics and biochemical tools. PtPSR belongs to a clade of Myb transcription factors that are conserved in stramenopiles and distantly related to plant P‐stress regulators. PtPSR bound specifically to a conserved cis‐regulatory element found in the regulatory region of P‐stress‐induced genes. ptpsr knockout mutants showed reduction in cell growth under P limitation. P‐stress responses were impaired in ptpsr mutants compared with wild‐type, including reduced induction of P‐stress response genes, near to complete loss of alkaline phosphatase activity and reduced phospholipid degradation. We conclude that PtPSR is a key transcription factor influencing P scavenging, phospholipid remodelling and cell growth in adaptation to P stress in diatoms.

The curation of genetic variants from biomedical articles is required for various clinical and research purposes. Nowadays, establishment of variant databases that include overall information about variants is becoming quite popular. These databases have immense utility, serving as a user-friendly information storehouse of variants for information seekers. While manual curation is the gold standard method for curation of variants, it can turn out to be time-consuming on a large scale thus necessitating the need for automation. Curation of variants described in biomedical literature may not be straightforward mainly due to various nomenclature and expression issues. Though current trends in paper writing on variants is inclined to the standard nomenclature such that variants can easily be retrieved, we have a massive store of variants in the literature that are present as non-standard names and the online search engines that are predominantly used may not be capable of finding them. For effective curation of variants, knowledge about the overall process of curation, nature and types of difficulties in curation, and ways to tackle the difficulties during the task are crucial. Only by effective curation, can variants be correctly interpreted. This paper presents the process and difficulties of curation of genetic variants with possible solutions and suggestions from our work experience in the field including literature support. The paper also highlights aspects of interpretation of genetic variants and the importance of writing papers on variants following standard and retrievable methods.

Adaptation is the central feature and leading explanation for the evolutionary diversification of life. Adaptation is also notoriously difficult to study in nature, owing to its complexity and logistically prohibitive timescale. Here, we leverage extensive contemporary and historical collections of Ambrosia artemisiifolia—an aggressively invasive weed and primary cause of pollen-induced hayfever—to track the phenotypic and genetic causes of recent local adaptation across its native and invasive ranges in North America and Europe, respectively. Large haploblocks—indicative of chromosomal inversions—contain a disproportionate share (26%) of genomic regions conferring parallel adaptation to local climates between ranges, are associated with rapidly adapting traits, and exhibit dramatic frequency shifts over space and time. These results highlight the importance of large-effect standing variants in rapid adaptation, which have been critical to A. artemisiifolia’s global spread across vast climatic gradients.

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