Predictable NHEJ Insertion and Assessment of HDR Editing Strategies in Plants.

Canonical CRISPR-Cas9 genome editing technique has profoundly impacted the fields of plant biology, biotechnology, and crop improvement. Since non-homologous end joining (NHEJ) is usually considered to generate random indels, its high efficiency mutation is generally not pertinent to precise editing. Homology-directed repair (HDR) can mediate precise editing with supplied donor DNA, but it suffers from extreme low efficiency in higher plants. Therefore, precision editing in plants will be facilitated by the ability to predict NHEJ repair outcome and to improve HDR efficiency. Here, we report that NHEJ-mediated single nucleotide insertion at different rice genes is predictable based on DNA sequences at the target loci. Three mutation prediction tools (inDelphi, FORECasT, and SPROUT) have been validated in the rice plant system. We also evaluated the chimeric guide RNA (cgRNA) and Cas9-Retron precISe Parallel Editing via homologY (CRISPEY) strategies to facilitate donor template supply for improving HDR efficiency in Nicotiana benthamiana and rice. However, neither cgRNA nor CRISPEY improved plant HDR editing efficiency in this study. Interestingly, our data indicate that tethering of 200-250 nucleotides long sequence to either 5' or 3' ends of guide RNA did not significantly affect Cas9 cleavage activity.

Single-nucleotide editing for zebra3 and wsl5 phenotypes in rice using CRISPR/Cas9-mediated adenine base editors.

The CRISPR/Cas9-mediated base editing technology can efficiently generate point mutations in the genome without introducing a double-strand break (DSB) or supplying a DNA donor template for homology-directed repair (HDR). In this study, adenine base editors (ABEs) were used for rapid generation of precise point mutations in two distinct genes, OsWSL5, and OsZEBRA3 (Z3), in both rice protoplasts and regenerated plants. The precisely engineered point mutations were stably inherited to subsequent generations. These single nucleotide alterations resulted in single amino acid changes and associated wsl5 and z3 phenotypes as evidenced by white stripe leaf and light green/dark green leaf pattern, respectively. Through selfing and genetic segregation, transgene-free, base edited wsl5 and z3 mutants were obtained in a short period of time. We noticed a novel mutation (V540A) in Z3 locus could also mimic the phenotype of Z3 mutation (S542P). Furthermore, we observed unexpected non- A/G or T/C mutations in the ABE editing window in a few of the edited plants. The ABE vectors and the method from this study could be used to simultaneously generate point mutations in multiple target genes in a single transformation and serve as a useful base editing tool for crop improvement as well as basic studies in plant biology.

Implementation and process analysis of pilot scale multi-phase anaerobic fermentation and digestion of faecal sludge in Ghana.

Background.  In Ghana, faecal sludge (FS) from on-site sanitation facilities is often discharged untreated into the environment, leading to significant insults to environmental and human health. Anaerobic digestion offers an attractive pathway for FS treatment with the concomitant production of energy in the form of methane. Another innovative option includes separating digestion into acidogenesis (production of volatile fatty acids (VFA)) and methanogenesis (production of methane), which could ultimately facilitate the production of an array of biofuels and biochemicals from the VFA. This work describes the development, implementation and modeling based analysis of a novel multiphase anaerobic fermentation-digestion process aimed at FS treatment in Kumasi, Ghana.  Methods.  A pilot-scale anaerobic fermentation process was implemented at the Kumasi Metropolitan Assembly's Oti Sanitary Landfill Site at Adanse Dompoase.  The process consisted of six 10 m reactors in series, which were inoculated with bovine rumen and fed with fecal sludge obtained from public toilets.  The performance of the fermentation process was characterized in terms of both aqueous and gaseous variables representing the conversion of influent organic carbon to VFA as well as CH 4.  Using the operating data, the first-ever process model for FS fermentation and digestion was developed and calibrated, based on the activated sludge model framework. Results and Conclusions.  This work represents one of the first systematic efforts at integrated FS characterization and process modeling to enable anaerobic fermentation and digestion of FS. It is shown that owing to pre-fermentation of FS in public septage holding tanks, one could employ significantly smaller digesters (lower capital costs) or increased loading capabilities for FS conversion to biogas or VFA. Further, using the first-ever calibrated process model for FS fermentation and digestion presented herein, we expect improved and more mechanistically informed development and application of different process designs and configurations for global FS management practice.

In vivo antioxidant activities of essential oils and their constituents from leaves of the Taiwanese Cinnamomum osmophloeum.

Cinnamomum osmophloeum Kaneh is an indigenous tree species in Taiwan. In this study, phytochemical characteristics and antioxidant activities of the essential oils and key constituents from the leaves of two C. osmophloeum clones were investigated. The two trees possess two chemotypes, which were classified as the cinnamaldehyde type and camphor type. We demonstrated that the essential oils from C. osmophloeum leaves exerted in vivo antioxidant activities in Caenorhabditis elegans. In addition, trans-cinnamaldehyde and D-(+)-camphor, which respectively represent the major compounds in the cinnamaldehyde-type and camphor-type trees, exerted significant in vivo antioxidant activities against juglone-induced oxidative stress in C. elegans. Moreover, expressions of antioxidative-related genes, including superoxide dismutase (SOD) and glutathione S-transferase (GST), were significantly induced by trans-cinnamaldehyde and D-(+)-camphor from C. osmophloeum leaves. Our results showed that the essential oils from C. osmophloeum leaves and their major compounds might have good potential for further development as nutraceuticals or antioxidant remedies.