Highly efficient multiplex editing: one-shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

Genome editing by RNA-guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene-free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T₁ and T₂, respectively). We developed novel counter-selection markers for N. benthamiana, most importantly Sl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on the production of toxic 5-fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY-UPP for selection of non-transgenic T₁, we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher-order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.     

Ingestion of colostrum from specific cows induces Bovine Neonatal Pancytopenia (BNP) in some calves

Background  

Since 2006, cases of haemorrhagic diathesis in young calves have been observed with a much higher incidence than previously known. The syndrome, now uniformly called Bovine Neonatal Pancytopenia (BNP), is characterized by multiple (external and internal) haemorrhages, thrombocytopenia, leukocytopenia, and bone marrow depletion. Although various infectious and toxicological causes of bleeding disorders in calves have been ruled out, the aetiology of BNP remains unknown. However, field observations have led to the hypothesis that the aetiological principle may be transmitted to calves via colostrum.     

Role of phenazine-enzyme physiology for current generation in a bioelectrochemical system

Pseudomonas aeruginosa produces phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO), which aid its anaerobic survival by mediating electron transfer to distant oxygen. These natural secondary metabolites are being explored in biotechnology to mediate electron transfer to the anode of bioelectrochemical systems. A major challenge is that only a small fraction of electrons from microbial substrate conversion is recovered. It remained unclear whether phenazines can re-enter the cell and thus, if the electrons accessed by the phenazines arise mainly from cytoplasmic or periplasmic pathways. Here, we prove that the periplasmic glucose dehydrogenase (Gcd) of P. aeruginosa and P. putida is involved in the reduction of natural phenazines. PYO displayed a 60-fold faster enzymatic reduction than PCA; PCA was, however, more stable for long-term electron shuttling to the anode. Evaluation of a Gcd knockout and overexpression strain showed that up to 9% of the anodic current can be designated to this enzymatic reaction. We further assessed phenazine uptake with the aid of two molecular biosensors, which experimentally confirm the phenazines’ ability to re-enter the cytoplasm. These findings significantly advance the understanding of the (electro) physiology of phenazines for future tailoring of phenazine electron discharge in biotechnological applications.     

Cypriniform fish in running waters reduce hyporheic oxygen depletion in a eutrophic river

1. The hyporheic zone is an important habitat for benthic invertebrates and early-developmental stages of gravel spawning fish. However, the eutrophication of running waters and, in turn, the excessive periphyton biomass leads to its biological clogging. The result of these processes is oxygen depletion and a reduction in the habitat quality of the hyporheic zone.
2. This study assessed whether top-down effects of two important European river fish species, the large herbivorous cypriniform common nase (Chondrostoma nasus, L.) and the large omnivorous cypriniform European chub (Squalius cephalus, L.), can reduce eutrophication effects in the hyporheic zone. A 4-week mesocosm-based field experiment in a eutrophic river was conducted using cage enclosures stocked or not with either nase or chub.
3. The top-down control of periphyton was expected to reduce biological clogging and thereby increase oxygen availability in the hyporheic zone. Accordingly, we hypothesised that in enclosures stocked with either fish the concentrations of dissolved oxygen in the hyporheic zone would be higher and the periphyton biomass would be lower than in enclosures without fish stocking.
4. Hyporheic oxygen concentrations were significantly higher in enclosures stocked with either nase or chub than in enclosures without fish stocking. However, periphyton ash-free dry mass was significantly reduced only in enclosures stocked with nase, not in those stocked with chub. Thus, the positive effects of nase and chub on hyporheic oxygen availability were caused by different mechanisms.
5. Our results demonstrate that nase and chub can reduce eutrophication effects in the hyporheic zone of running waters. Hence, protecting and enhancing stocks of herbivorous and omnivorous fish will contribute to restoring the hyporheic zone in efforts to preserve biodiversity in eutrophic rivers.

     

Motivation Matters: Lessons for REDD+ Participatory Measurement,Reporting and Verification from Three Decades of Child Health Participatory Monitoring in Indonesia

Participatory Measurement, Reporting and Verification (PMRV), in the context of reducing emissions from deforestation and forest degradation with its co-benefits (REDD+) requires sustained monitoring and reporting by community members. This requirement appears challenging and has yet to be achieved. Other successful, long established, community self-monitoring and reporting systems may provide valuable lessons. The Indonesian integrated village healthcare program (Posyandu) was initiated in the 1980s and still provides effective and successful participatory measurement and reporting of child health status across the diverse, and often remote, communities of Indonesia. Posyandu activities focus on the growth and development of children under the age of five by recording their height and weight and reporting these monthly to the Ministry of Health. Here we focus on the local Posyandu personnel (kaders) and their motivations and incentives for contributing. While Posyandu and REDD+ measurement and reporting activities differ, there are sufficient commonalities to draw useful lessons. We find that the Posyandu kaders are motivated by their interests in health care, by their belief that it benefits the community, and by encouragement by local leaders. Recognition from the community, status within the system, training opportunities, competition among communities, and small payments provide incentives to sustain participation. We examine these lessons in the context of REDD+.     

Phylogenetic analysis ofEremosyne pectinata (Saxifragaceae s. l.) based onrbcL sequence data

Earlier analyses ofrbcL sequences clarified phylogenetic relationships of 16 of 17 subfamilies ofSaxifragaceae s. l. This study investigates the affinities of the monotypicEremosynoideae, the only subfamily ofSaxifragaceae s. l. not examined previously forrbcL sequence variation. Our analyses suggest thatEremosyne is only distantly related to core members ofSaxifragaceae s. l. (i.e.Saxifragoideae = Saxifragaceae s. str.); it is allied instead with members ofAsteridae. A particularly close relationship is indicated betweenEremosyne andEscallonia, another traditional member ofSaxifragaceae s. l. that also appears as part ofAsteridae. Inclusion ofEremosyne withinAsteridae is in agreement with embryology, as well as with 18S rDNA sequence data.