Summary The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9‐induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9‐induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large‐scale off‐targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off‐target sites of a large number of T0 plants, low‐frequency mutations were found in only one off‐target site where the sequence had 1‐bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering.

Αggregation of phenylacetylene macrocycles (PAMs) in solution has been studied by 1H NMR spectroscopy and vapor pressure osmometry. The association constant for dimerization, K assoc, has been determined by curve fitting the concentration dependence of 1H NMR chemical shifts to a model for monomer−dimer equilibrium. The reliability of the NMR-determined aggregation constants and aggregate size have been independently verified by vapor pressure osmometry measurements. Thermodynamic parameters for association have been obtained from van't Hoff analyses which show the aggregation to be favored enthalpically. The aggregation of PAMs bearing various endo- and exo-annular functional groups and PAMs of different geometry and ring size has been studied. The type of pendant functional groups and the manner in which these groups are arranged on the macrocycle is shown to strongly influence self-association. PAMs substituted with electron withdrawing groups (e.g., esters) are more strongly associated than those bearing electron donating groups (e.g., alkyl ethers) or macrocycles bearing a combination of the two substituents. The type of alkyl substituent on the ester or ether group is less important as long as the substituent is not branched and is exo-annular. Endo-annular alkyl ethers as well as branched exo-annular alkyl esters severely disrupt aggregation. Rigidity of the macrocycle also influences self-association. In contrast to hexameric macrocycles, similarly substituted open-chain oligomers and a nonplanar macrocycle show much weaker association. These findings are discussed in the context of face-to-face π−π interactions between aromatic rings. Consideration has also been given to π−π interactions between aromatic and ethynyl groups and between a pair of acetylenes, but these are concluded to be less significant based on an analysis of data from the Cambridge Structural Database.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat. Here we report a 110-Mb draft sequence of Pst isolate CY32, obtained using a ‘fosmid-to-fosmid’ strategy, to better understand its race evolution and pathogenesis. The Pst genome is highly heterozygous and contains 25,288 protein-coding genes. Compared with non-obligate fungal pathogens, Pst has a more diverse gene composition and more genes encoding secreted proteins. Re-sequencing analysis indicates significant genetic variation among six isolates collected from different continents. Approximately 35% of SNPs are in the coding sequence regions, and half of them are non-synonymous. High genetic diversity in Pst suggests that sexual reproduction has an important role in the origin of different regional races. Our results show the effectiveness of the ‘fosmid-to-fosmid’ strategy for sequencing dikaryotic genomes and the feasibility of genome analysis to understand race evolution in Pst and other obligate pathogens.

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A convergent, stepwise synthesis of linear phenylacetylene sequences (PASs) is described. The methodology allows for complete control over chain length, sequence order of monomers, and functional group placement. Chain growth follows geometric progression thus allowing sequences of length 2", where n is the number of repetitive cycles, to be assembled in a total of just 3.n steps (two deprotections and one coupling for each cycle). Sequences of length other than 2" as well as sequences having a particular arrangement of co-monomer units, can also be realized by merging parallel repetitive cycles. Upon deprotection of the termini, these PASs can be cyclized to phenylacetylene macrocycles (PAMs) in high yield. Control over the ring structure of PAMs is determined by the chemistry of precursor PASs; the size of the macrocycle is related to the sequence length, while the geometry of the macrocycle and the position of the pendant functional groups on the macrocycle is governed by co-monomer sequence order. PAMs with four, five, six, seven, and twelve phenylacetylene monomer units, as well as a variety of site-specifically-functionalized PAMs, have been synthesized with this method. Finally, functional group transformations have been performed on some of the PAMs which lead to PAMs with new functionality. Theversatileand efficient approach to this family of geometrically well-defined macrocycles offers potential for producing a set of modular building blocks to rationally assemble molecular crystals and liquid crystals. For this reason, the solid-state characteristics of the hydrocarbon skeletons are of interest. In spite of their solubility in common solvents, hydrocarbon PAMs are shown to yield crystals with remarkable thermal stability and high melting points. Three PAM hydrocarbons are shown not to exhibit melting transitions up to ca 400 OC, at which point an abrupt thermal irreversible reaction occurs, apparently involving a solid-state polymerization of the acetylene units.

By fusing the extracellular domain of the natural killer (NK) cell receptor NKG2D to DAP12, we constructed a chimeric antigen receptor (CAR) to improve NK cell tumor responses. An RNA electroporation approach that provides transient expression of the CAR was adopted as a risk mitigation strategy. Expression of the NKG2D RNA CAR significantly augmented the cytolytic activity of NK cells against several solid tumor cell lines in vitro and provided a clear therapeutic benefit to mice with established solid tumors. Three patients with metastatic colorectal cancer were then treated with local infusion of the CAR-NK cells. Reduction of ascites generation and a marked decrease in number of tumor cells in ascites samples were observed in the first two patients treated with intraperitoneal infusion of low doses of the CAR-NK cells. The third patient with metastatic tumor sites in the liver was treated with ultrasound-guided percutaneous injection, followed by intraperitoneal infusion of the CAR-NK cells. Rapid tumor regression in the liver region was observed with Doppler ultrasound imaging and complete metabolic response in the treated liver lesions was confirmed by positron emission tomography (PET)-computed tomographic (CT) scanning. Our results highlight a promising therapeutic potential of using RNA CAR-modified NK cells to treat metastatic colorectal cancer.

MicroRNAs are 19- to 22-nucleotide small noncoding RNAs that have been implicated in abiotic stress responses. In this study, we found that knockdown of microRNA166, using the Short Tandem Target Mimic (STTM) system, resulted in morphological changes that confer drought resistance in rice (). From a large-scale screen for miRNA knockdown lines in rice, we identified miR166 knockdown lines (STTM166); these plants exhibit a rolled-leaf phenotype, which is normally displayed by rice plants under drought stress. The leaves of STTM166 rice plants had smaller bulliform cells and abnormal sclerenchymatous cells, likely causing the rolled-leaf phenotype. The STTM166 plants had reduced stomatal conductance and showed decreased transpiration rates. The STTM166 lines also exhibited altered stem xylem and decreased hydraulic conductivity, likely due to the reduced diameter of the xylem vessels. Molecular analyses identified rice (), a member of HD-Zip III gene family, as a major target of miR166; moreover, rice plants overexpressing a miR166-resistant form of resembled the STTM166 plants, including leaf rolling and higher drought resistance. The genes downstream of miR166- consisted of polysaccharide synthesis-related genes that may contribute to cell wall formation and vascular development. Our results suggest that drought resistance in rice can be increased by manipulating miRNAs, which leads to developmental changes, such as leaf rolling and reduced diameter of the xylem, that mimic plants' natural responses to water-deficit stress.

Improvements in plant agricultural productivity are urgently needed to reduce the dependency on limited natural resources and produce enough food for a growing world population. Human intervention over thousands of years has improved the yield of important crops; however, it is increasingly difficult to find new targets for genetic improvement. MicroRNAs (miRNAs) are promising targets for crop improvement, but their inactivation is technically challenging and has hampered functional analyses. We have produced a large collection of transgenic short tandem target mimic (STTM) lines silencing 35 miRNA families in rice as a resource for functional studies and crop improvement. Visual assessment of field-grown miRNA-silenced lines uncovered alterations in many valuable agronomic traits, including plant height, tiller number, and grain number, that remained stable for up to five generations. We show that manipulation of miR398 can increase panicle length, grain number, and grain size in rice. In addition, we discovered additional agronomic functions for several known miRNAs, including miR172 and miR156. Our collection of STTM lines thus represents a valuable resource for functional analysis of rice miRNAs, as well as for agronomic improvement that can be readily transferred to other important food crops.small RNA | microRNA | silencing | yield | crop