Efficient CRISPR/Cas9-based genome editing in carrot cells

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) is a powerful genome editing tool that has been widely adopted in model organisms recently, but has not been used in carrot—a model species for in vitro culture studies and an important health-promoting crop grown worldwide. In this study, for the first time, we report application of the CRISPR/Cas9 system for efficient targeted mutagenesis of the carrot genome. Multiplexing CRISPR/Cas9 vectors expressing two single-guide RNA (gRNAs) targeting the carrot flavanone-3-hydroxylase (F3H) gene were tested for blockage of the anthocyanin biosynthesis in a model purple-colored callus using Agrobacterium-mediated genetic transformation. This approach allowed fast and visual comparison of three codon-optimized Cas9 genes and revealed that the most efficient one in generating F3H mutants was the Arabidopsis codon-optimized AteCas9 gene with up to 90% efficiency. Knockout of F3H gene resulted in the discoloration of calli, validating the functional role of this gene in the anthocyanin biosynthesis in carrot as well as providing a visual marker for screening successfully edited events. Most resulting mutations were small Indels, but long chromosome fragment deletions of 116–119 nt were also generated with simultaneous cleavage mediated by two gRNAs. The results demonstrate successful site-directed mutagenesis in carrot with CRISPR/Cas9 and the usefulness of a model callus culture to validate genome editing systems. Given that the carrot genome has been sequenced recently, our timely study sheds light on the promising application of genome editing tools for boosting basic and translational research in this important vegetable crop

X-ray absorption spectroscopy study of diluted magnetic semiconductors: Zn1-xMxSe (M = Mn, Fe, Co) and Zn1-xMnxY (Y = Se, Te)

We have investigated 3d electronic states of doped transition metals in II-VI diluted magnetic semiconductors, Zn1-xMxSe (M = Mn, Fe, Co) and Zn1-xMnxY (Y = Se, Te), using the transition-metal L2,3-edge X-ray absorption spectroscopy (XAS) measurements. In order to explain the XAS spectra, we employed a tetragonal cluster model calculation, which includes not only the full ionic multiplet structure but also configuration interaction (CI). The results show that CI is essential to describe the experimental spectra adequately, indicating the strong hybridization between the transition metal 3d and the ligand p orbitals. In the study of Zn1-xMnxY (Y = Se, Te), we also found considerable spectral change in the Mn L2,3-edge XAS spectra for different ligands, confirming the importance of the hybridization effects in these materials.Comment: This paper consists of 22 pages including 4 figures. This paper is submitted to Physical Review

Role of nickel in high rate methanol degradation in anaerobic granular sludge bioreactors

The effect of nickel deprivation from the influent of a mesophilic (30°C) methanol fed upflow anaerobic sludge bed (UASB) reactor was investigated by coupling the reactor performance to the evolution of the Methanosarcina population of the bioreactor sludge. The reactor was operated at pH 7.0 and an organic loading rate (OLR) of 5–15 g COD l−1 day−1 for 191 days. A clear limitation of the specific methanogenic activity (SMA) on methanol due to the absence of nickel was observed after 129 days of bioreactor operation: the SMA of the sludge in medium with the complete trace metal solution except nickel amounted to 1.164 (±0.167) g CH4-COD g VSS−1 day−1 compared to 2.027 (±0.111) g CH4-COD g VSS−1 day−1 in a medium with the complete (including nickel) trace metal solution. The methanol removal efficiency during these 129 days was 99%, no volatile fatty acid (VFA) accumulation was observed and the size of the Methanosarcina population increased compared to the seed sludge. Continuation of the UASB reactor operation with the nickel limited sludge lead to incomplete methanol removal, and thus methanol accumulation in the reactor effluent from day 142 onwards. This methanol accumulation subsequently induced an increase of the acetogenic activity in the UASB reactor on day 160. On day 165, 77% of the methanol fed to the system was converted to acetate and the Methanosarcina population size had substantially decreased. Inclusion of 0.5 μM Ni (dosed as NiCl2) to the influent from day 165 onwards lead to the recovery of the methanol removal efficiency to 99% without VFA accumulation within 2 days of bioreactor operation

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Methanogens, sulphate and heavy metals: a complex system

Anaerobic digestion (AD) is a well-established technology used for the treatment of wastes and wastewaters with high organic content. During AD organic matter is converted stepwise to methane-containing biogasa renewable energy carrier. Methane production occurs in the last AD step and relies on methanogens, which are rather sensitive to some contaminants commonly found in wastewaters (e.g. heavy metals), or easily outcompeted by other groups of microorganisms (e.g. sulphate reducing bacteria, SRB). This review gives an overview of previous research and pilot-scale studies that shed some light on the effects of sulphate and heavy metals on methanogenesis. Despite the numerous studies on this subject, comparison is not always possible due to differences in the experimental conditions used and parameters explained. An overview of the possible benefits of methanogens and SRB co-habitation is also covered. Small amounts of sulphide produced by SRB can precipitate with metals, neutralising the negative effects of sulphide accumulation and free heavy metals on methanogenesis. Knowledge on how to untangle and balance sulphate reduction and methanogenesis is crucial to take advantage of the potential for the utilisation of biogenic sulphide as a metal detoxification agent with minimal loss in methane production in anaerobic digesters.The research was financially supported by the People Program (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007-2013 under REA agreement 289193