SMAP design: a multiplex PCR amplicon and gRNA design tool to screen for natural and CRISPR-induced genetic variation.

Multiplex amplicon sequencing is a versatile method to identify genetic variation in natural or mutagenized populations through eco-tilling or multiplex CRISPR screens. Such genotyping screens require reliable and specific primer designs, combined with simultaneous gRNA design for CRISPR screens. Unfortunately, current tools are unable to combine multiplex gRNA and primer design in a high-throughput and easy-to-use manner with high design flexibility. Here, we report the development of a bioinformatics tool called SMAP design to overcome these limitations. We tested SMAP design on several plant and non-plant genomes and obtained designs for more than 80-90% of the target genes, depending on the genome and gene family. We validated the designs with Illumina multiplex amplicon sequencing and Sanger sequencing in Arabidopsis, soybean, and maize. We also used SMAP design to perform eco-tilling by tilling PCR amplicons across nine candidate genes putatively associated with haploid induction in Cichorium intybus. We screened 60 accessions of chicory and witloof and identified thirteen knockout haplotypes and their carriers. SMAP design is an easy-to-use command-line tool that generates highly specific gRNA and/or primer designs for any number of loci for CRISPR or natural variation screens and is compatible with other SMAP modules for seamless downstream analysis.

A rapid and multi-endpoint ecotoxicological test using Mychonastes afer for efficient screening of metals and herbicides.

Microalgal growth-based tests are international standards for ecotoxicity assessment; however, their long exposure times, large sample volumes, and reliance on a single growth-endpoint make them inadequate for rapid toxicity screening. Here, we aimed to develop a rapid and simple ecotoxicological test using the fast-growing green alga Mychonastes afer, with multiple endpoints-growth, lipid content, and photosynthesis. We exposed M. afer to two metals-silver and copper-and two herbicides-atrazine and diuron-for 24 h and identified the most sensitive and reliable endpoints for each toxicant: the maximum electron transport rate (ETRmax) for Ag, Cu and atrazine, and the lipid content for diuron. Lipid content was found to be both a sensitive and reliable biomarker, meeting the effluent limit guidelines in both the Republic of Korea and the USA. The sensitivity of M. afer to Ag and atrazine also closely matched the HC5 values derived from the species sensitivity distribution approach, confirming its reliability for setting regulatory concentrations of these contaminants. Our calculated predicted no-effect concentration (PNEC) values were similar to established European Union PNECs for Ag, Cu, atrazine, and diuron, underlining the utility of these biological endpoints for ecological risk assessment and regulatory decision making. This method required lower sample volume (2 mL vs 100 mL) and exposure time (24 h vs 72-120 h) than conventional green algal tests, and eliminated the need for labour-intensive cell counting, expensive equipment, and chlorophyll fluorescence measurement expertise. Overall, this M. afer test can be a valuable tool for the rapid screening of wastewater for metals and herbicides, contributing to environmental protection and management practices.

Testing the toxicity of metals, phenol, effluents, and receiving waters by root elongation in Lactuca sativa L.

Phytotoxicity tests using higher plants are among the most simple, sensitive, and cost-effective of the methods available for ecotoxicity testing. In the present study, a hydroponic-based phytotoxicity test using seeds of Lactuca sativa was used to evaluate the water quality of receiving waters and effluents near two industrial sites (Soyo and Daejon) in Korea with respect to the toxicity of 10 metals (As, Cd, Cr, Cu, Fe, Pb, Mn, Hg, Ni, Zn) and phenol, and of the receiving waters and effluents themselves. First, the L. sativa hydroponic bioassay was used to determine whether the receiving water or effluents were toxic; then, the responsible toxicant was identified. The results obtained with the L. sativa bioassay ranked the EC50 toxicities of the investigated metal ions and phenol as: Cd > Ni > Cu > Zn > Hg > phenol > As > Mn > Cr > Pb > Fe. We found that Zn was the toxicant principally responsible for toxicity in Daejeon effluents. The Daejeon field effluent had a higher Zn concentration than permitted by the effluent discharge criteria of the Ministry of Environment of Korea. Our conclusion on the importance of Zn toxicity was supported by the results of the L. sativa hydroponic assay, which showed that the concentration of Zn required to inhibit root elongation in L. sativa by 50% (EC50) was higher in the Daejeon field effluent than that of pure Zn. More importantly, we proved that the L. sativa hydroponic test method can be applied not only as an alternative tool for determining whether a given waste is acceptable for discharge into public water bodies, but also as an alternative method for measuring the safety of aquatic environments using EC20 values, with respect to the water pollutants investigated (i.e., Cd, Cr, Cu, Pb, Mn, Hg, Ni, Zn, and phenol).

Genome editing in macroalgae: advances and challenges.

This minireview examines the current state and challenges of genome editing in macroalgae. Despite the ecological and economic significance of this group of organisms, genome editing has seen limited applications. While CRISPR functionality has been established in two brown (Ectocarpus species 7 and Saccharina japonica) and one green seaweed (Ulva prolifera), these studies are limited to proof-of-concept demonstrations. All studies also (co)-targeted ADENINE PHOSPHORIBOSYL TRANSFERASE to enrich for mutants, due to the relatively low editing efficiencies. To advance the field, there should be a focus on advancing auxiliary technologies, particularly stable transformation, so that novel editing reagents can be screened for their efficiency. More work is also needed on understanding DNA repair in these organisms, as this is tightly linked with the editing outcomes. Developing efficient genome editing tools for macroalgae will unlock the ability to characterize their genes, which is largely uncharted terrain. Moreover, given their economic importance, genome editing will also impact breeding campaigns to develop strains that have better yields, produce more commercially valuable compounds, and show improved resilience to the impacts of global change.

Interactive Effects of Blue Light and Water Turbulence on the Growth of the Green Macroalga Ulva australis (Chlorophyta).

Macroalgal growth and yield are key to sustainable aquaculture. Although light and water turbulence are two important factors that affect algal productivity, research on their interaction is limited. Therefore, in this study, we investigated the effects of different wavelengths of light and the presence or absence of water turbulence on the growth of the green macroalga Ulva australis. Water turbulence was found to enhance the growth of U. australis irrespective of photosynthetic performance, but only in blue light cultures. The quantum dose of blue light required to induce 50% growth promotion was 1.02 mol m-2, which is comparable to the reported values for cryptochrome-mediated effects in other macroalgae. The combined effect of blue light and water turbulence led to the accumulation of photosynthesis-related proteins that support plastid differentiation and facilitate efficient photosynthesis and growth. Our findings thus highlight the potential of harnessing blue light and water turbulence to maximise macroalgal cultivation for sustainable and profitable algal aquaculture.

Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts.

Site-specific recombinases such as the Cre-LoxP system are routinely used for genome engineering in both prokaryotes and eukaryotes. Importantly, recombinases complement the CRISPR-Cas toolbox and provide the additional benefit of high-efficiency DNA editing without generating toxic DNA double-strand breaks, allowing multiple recombination events at the same time. However, only a handful of independent, orthogonal recombination systems are available, limiting their use in more complex applications that require multiple specific recombination events, such as metabolic engineering and genetic circuits. To address this shortcoming, we develop 63 symmetrical LoxP variants and test 1192 pairwise combinations to determine their cross-reactivity and specificity upon Cre activation. Ultimately, we establish a set of 16 orthogonal LoxPsym variants and demonstrate their use for multiplexed genome engineering in both prokaryotes (E. coli) and eukaryotes (S. cerevisiae and Z. mays). Together, this work yields a significant expansion of the Cre-LoxP toolbox for genome editing, metabolic engineering and other controlled recombination events, and provides insights into the Cre-LoxP recombination process.

Giant Duckweed (Spirodela polyrhiza) Root Growth as a Simple and Sensitive Indicator of Copper and Chromium Contamination.

Aquatic environment are often contaminated with heavy metals from various industrial sources. However, physicochemical techniques for pollutant detection are limited, thus prompting the need for additional bioassays. We investigated the use of greater duckweed (Spirodela polyrhiza) as a bioindicator of metal pollution. We exposed S. polyrhiza to four pollutants (namely, silver, cadmium, copper, and chromium) and assessed metal toxicity by measuring its frond area and the length of its regrown roots. The plant displayed significant differences in both frond size and root growth in response to the four metals. Silver was the most toxic (EC50 = 23 µg L-1) while copper the least (EC50 = 365-607 µg L-1). Direct comparisons of metal sensitivity and the reliability of the two endpoint assays showed that root growth was more sensitive (lower in terms of 50% effective concentration) to chromium, cadmium, and copper, and was more reliable (lower in terms of coefficient of variation) than those for frond area. Compared to conventional Lemna-based tests, the S. polyrhiza test is easier to perform (requiring only one 24-well plate, 3 mL of medium and a 72-h exposure). Moreover, it does not require livestock cultivation/maintenance, making it more suitable for repeated measurements. Measurements of S. polyrhiza root length may be suitable for assessment when copper and chromium in municipal and industrial wastewater exceed the environmentally permissible levels.

Evaluating ecotoxicological assays for comprehensive risk assessment of toxic metals present in industrial wastewaters in the Republic of Korea.

Toxicity tests represent a rapid, user-friendly and cost-effective means to assess the impact of wastewater quality on aquatic ecosystems. There are not many cases where wastewater management standards are set based on various bio-based ecotoxicity values. Here, we tested a novel multitaxon approach to compare standard water quality indices to toxicity metrics obtained from ecotoxicity tests, conducted using aquatic organisms representing several trophic levels (Aliivibrio, Ulva, Daphnia, and Lemna), for 99 industrial wastewater samples from South Korea. For five wastewater samples, the concentrations of Se, Zn, or Ni exceeded the permissible limits (1, 5, and 3 mg L-1, respectively). All the four physiochemical water quality indices tested were positively correlated with Se and Pb concentrations. The toxicity unit (TU) scores indicated a declining sensitivity to pollutants, in the order Lemna (2.87) >Daphnia (2.24) >Aliivibrio (1.78) >Ulva (1.42). Significant correlations were observed between (1) Cd and Ni, and Aliivibrio, (2) Cu and Daphnia, (3) Cd, Cu, Zn, and Cr and Lemna, and (4) Cu, Zn, and Ni and Ulva. Daphnia-Lemna and Lemna-Ulva were found to be good indicators of ecologically harmful Se and Ni contents in wastewater, respectively. We suggest that regulatory thresholds based on these bioassays should be set at TU = 1 for all the species or at TU = 1 for Aliivibrio and Ulva and TU = 2 for Daphnia and Lemna, if the number of companies whose wastewater discharge exceeds the allowable TU levels is <1 % or 5 % of the total number of industries, respectively. Taken together, these findings could help in establishing a rapid, ecologically relevant wastewater quality assessment system that would be useful for developing strategies to protect aquatic ecosystems.

Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform.

BACKGROUND: Testing an ever-increasing number of CRISPR components is challenging when developing new genome engineering tools. Plant biotechnology has few high-throughput options to perform iterative design-build-test-learn cycles of gene-editing reagents. To bridge this gap, we develop ITER (Iterative Testing of Editing Reagents) based on 96-well arrayed protoplast transfections and high-content imaging. RESULTS: We validate ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors (ABEs), allowing one optimization cycle - from design to results - within 3 weeks. Given that previous LbCas12a-ABEs have low or no activity in plants, we use ITER to develop an optimized LbCas12a-ABE. We show that sequential improvement of five components - NLS, crRNA, LbCas12a, adenine deaminase, and linker - leads to a remarkable increase in activity from almost undetectable levels to 40% on an extrachromosomal GFP reporter. We confirm the activity of LbCas12a-ABE at endogenous targets in protoplasts and obtain base-edited plants in up to 55% of stable wheat transformants and the edits are transmitted to T1 progeny. We leverage these improvements to develop a highly mutagenic LbCas12a nuclease and a LbCas12a-CBE demonstrating that the optimizations can be broadly applied to the Cas12a toolbox. CONCLUSION: Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants. We use ITER to create an efficient Cas12a-ABE by iteratively testing a large panel of vector components. ITER will likely be useful to create and optimize genome editing reagents in a wide range of plant species.

Simple and Efficient Modification of Golden Gate Design Standards and Parts Using Oligo Stitching.

The assembly of DNA parts is a critical aspect of contemporary biological research. Gibson assembly and Golden Gate cloning are two popular options. Here, we explore the use of single stranded DNA oligos with Gibson assembly to augment Golden Gate cloning workflows in a process called "oligo stitching". Our results show that oligo stitching can efficiently convert Golden Gate parts between different assembly standards and directly assemble incompatible Golden Gate parts without PCR amplification. Building on previous reports, we show that it can also be used to assemble de novo sequences. As a final application, we show that restriction enzyme recognition sites can be removed from plasmids and utilize the same concept to perform saturation mutagenesis. Given oligo stitching's versatility and high efficiency, we expect that it will be a useful addition to the molecular biologist's toolbox.

IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction.

BACKGROUND: Copy number determination is one of the first steps in the characterization of transgenic plant lines. The classical approach to this, Southern blotting, is time-consuming, expensive and requires massive amounts of high-quality genomic DNA. Other PCR-based techniques are either inaccurate, laborious, or expensive. RESULTS: Here, we propose a new technique, IMPLANT (Insertion of competitive PCR calibrator for copy number estimation), a competitive PCR-based technique in which the competitor (based on an endogenous gene) is also incorporated in the T-DNA, which then gets integrated in the genome together with the gene of interest. As the number of integrated competitor molecules directly corresponds to the number of transgene copies, the transgene copy number can be determined by a single PCR reaction. We demonstrate that the results of this technique closely correspond with those obtained by segregation analysis in Arabidopsis and digital PCR In rice, indicating that it is a powerful alternative for other techniques for copy number determination. CONCLUSIONS: We show that this technique is not only reliable, but is also faster, easier, and cheaper as compared with other techniques. Accurate results are obtained in both Arabidopsis and rice, but this technique can be easily extended to other organisms and as such can be widely adopted in the field of biotechnology.

Commercial Potential of the Cyanobacterium Arthrospira maxima: Physiological and Biochemical Traits and the Purification of Phycocyanin.

Arthrospira maxima is a natural source of fine chemicals for multiple biotechnological applications. We determined the optimal environmental conditions for A. maxima by measuring its relative growth rate (RGR), pigment yield, and photosynthetic performance under different pH and temperature conditions. RGR was highest at pH 7-9 and 30 °C. Chlorophyll a, phycocyanin, maximal quantum yield (Fv/Fm), relative maximal electron transport rate (rETRmax), and effective quantum yield (ΦPSII) were highest at pH 7-8 and 25 °C. Interestingly, phycoerythrin and allophycocyanin content was highest at 15 °C, which may be the lowest optimum temperature reported for phycobiliprotein production in the Arthrospira species. A threestep purification of phycocyanin (PC) by ultrafiltration, ion-exchange chromatography, and gel filtration resulted in a 97.6% purity of PC.

Agrobacterium strains and strain improvement: Present and outlook.

Almost 40 years ago the first transgenic plant was generated through Agrobacterium tumefaciens-mediated transformation, which, until now, remains the method of choice for gene delivery into plants. Ever since, optimized Agrobacterium strains have been developed with additional (genetic) modifications that were mostly aimed at enhancing the transformation efficiency, although an optimized strain also exists that reduces unwanted plasmid recombination. As a result, a collection of very useful strains has been created to transform a wide variety of plant species, but has also led to a confusing Agrobacterium strain nomenclature. The latter is often misleading for choosing the best-suited strain for one's transformation purposes. To overcome this issue, we provide a complete overview of the strain classification. We also indicate different strain modifications and their purposes, as well as the obtained results with regard to the transformation process sensu largo. Furthermore, we propose additional improvements of the Agrobacterium-mediated transformation process and consider several worthwhile modifications, for instance, by circumventing a defense response in planta. In this regard, we will discuss pattern-triggered immunity, pathogen-associated molecular pattern detection, hormone homeostasis and signaling, and reactive oxygen species in relationship to Agrobacterium transformation. We will also explore alterations that increase agrobacterial transformation efficiency, reduce plasmid recombination, and improve biocontainment. Finally, we recommend the use of a modular system to best utilize the available knowledge for successful plant transformation.

Application of a programmed semi-automated Ulva pertusa bioassay for testing single toxicants and stream water quality.

A toxicity test based on inhibition of reproduction in the green macroalga Ulva pertusa involves quantifying the change in thallus color as reproduction progresses. However, interpretation of this color change is reliant on the skill level of the examiner. This study aimed to validate a new toxicity test based on inhibition of reproduction in the green macroalga U. pertusa using a vital stain and programmed semi-automated analysis (using Image J) of the change in thallus color. The toxicity rank by inverse EC50 values was: irgarol (0.048 mg L-1) > Ag (0.132 mg L-1) > As (0.172 mg L-1) > simazine (0.378 mg L-1) > formaldehyde (0.442 mg L-1) > DCOIT (0.783 mg L-1) > ZnPT (3.556 mg L-1) > medetomidine (11.600 mg L-1) > phenol (29.316 mg L-1) > methanol (2,736 mg L-1) > ethanol (3,306 mg L-1). The sensitivity of the U. pertusa test to stream waters was similar to or lower than those of the commonly-used Lemna minor and Daphnia magna bioassays. The U. pertusa bioassay is sensitive to, and suitable for, testing various toxicants including metals, volatile organic compounds, herbicide, antifouling agents and phenol and can also be applied to testing freshwater quality after salinity adjustment.

Reappraisal of the toxicity test method using the green alga Ulva pertusa Kjellman (Chlorophyta).

This study was aimed to develop an objective way of quantifying the reproductive status of the green macroalga, Ulva pertusa using a vital stain and programmed automated analysis (by Image J program). The EC50 values (with 95% CI), the concentrations of toxicants inducing a reduction of 50% in sporulation after 96 h exposure, from the newly developed method were similar to those obtained by the conventional method: 0.651 (0.598-0.705) mg l-1 for Cd, 0.144 (0.110-0.162) mg l-1 for Cu, 0.180 (0.165-0.195) mg l-1 for atrazine, 0.076 (0.049-0.094) mg l-1 for diuron and 30.6 (26.5-34.4) ml l-1 for DMSO, respectively. When the EC50 values from this study were compared to that those from literatures, the sensitivity for some toxicants was similar or higher than that of U. fasciata (1.930 mg l-1 for germination for Cd), U. armoricana (0.250 mg l-1 for Fv/Fm for Cu), U. reticulata (0.126-1.585 mg l-1 for growth for Cu), and U. intestinalis (0.650 mg l-1 for Fv/Fm for atrazine). The subjective views of the experimental performers can be eliminated using the newly developed method. The Ulva method gave consistent responses to Cu and Cd of internationally allowable ranges for effluents, implying that the method is a useful tool for monitoring industrial wastewaters containing these metals.