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Precision DNA/gene replacement is a promising genome-editing tool that is highly desirable for molecular engineering and breeding by design. Although the CRISPR/Cas9 system works well as a tool for gene knockout in plants, gene replacement has rarely been reported. Towards this end, we first designed a combinatory dual-sgRNA/Cas9 vector (construct #1) that successfully deleted miRNA gene regions (MIR169a and MIR827a). The deletions were confirmed by PCR and subsequent sequencing, yielding deletion efficiencies of 20% and 24% on MIR169a and MIR827a loci, respectively. We designed a second structure (construct #2) that contains sites homologous to Arabidopsis TERMINAL FLOWER 1 (TFL1) for homology-directed repair (HDR) with regions corresponding to the two sgRNAs on the modified construct #1. The two constructs were co-transformed into Arabidopsis plants to provide both targeted deletion and donor repair for targeted gene replacement by HDR. Four of 500 stably transformed T0 transgenic plants (0.8%) contained replaced fragments. The presence of the expected recombination sites was further confirmed by sequencing. Therefore, we successfully established a gene deletion/replacement system in stably transformed plants that can potentially be utilized to introduce genes of interest for targeted crop improvement.

BackgroundThe harvest index for many crops can be improved through introduction of dwarf stature to increase lodging resistance, combined with early maturity. The inbred line Shen5003 has been widely used in maize breeding in China as a key donor line for the dwarf trait. Also, one major quantitative trait locus (QTL) controlling plant height has been identified in bin 5.05–5.06, across several maize bi-parental populations. With the progress of publicly available maize genome sequence, the objective of this work was to identify the candidate genes that affect plant height among Chinese maize inbred lines with genome wide association studies (GWAS).Methods and FindingsA total of 284 maize inbred lines were genotyped using over 55,000 evenly spaced SNPs, from which a set of 41,101 SNPs were filtered with stringent quality control for further data analysis. With the population structure controlled in a mixed linear model (MLM) implemented with the software TASSEL, we carried out a genome-wide association study (GWAS) for plant height. A total of 204 SNPs (P≤0.0001) and 105 genomic loci harboring coding regions were identified. Four loci containing genes associated with gibberellin (GA), auxin, and epigenetic pathways may be involved in natural variation that led to a dwarf phenotype in elite maize inbred lines. Among them, a favorable allele for dwarfing on chromosome 5 (SNP PZE-105115518) was also identified in six Shen5003 derivatives.ConclusionsThe fact that a large number of previously identified dwarf genes are missing from our study highlights the discovery of the consistently significant association of the gene harboring the SNP PZE-105115518 with plant height (P = 8.91e-10) and its confirmation in the Shen5003 introgression lines. Results from this study suggest that, in the maize breeding schema in China, specific alleles were selected, that have played important roles in maize production.

With the goal of increasing the complexity of metallo-supramolecules, two rhombus star-shaped supramolecular architectures, namely, supersnowflakes, were designed and assembled using multiple 2,2':6',2″-terpyridine (tpy) ligands in a stepwise manner. In the design of multicomponent self-assembly, ditopic and tritopic ligands were bridged through Ru(II) with strong coordination to form metal-organic ligands for the subsequent self-assembly with a hexatopic ligand and Zn(II). The combination of Ru(II)-organic ligands with high stability and Zn(II) ions with weak coordination played a key role in the self-assembly of giant heteroleptic supersnowflakes, which encompassed three types of tpy-based organic ligands and two metal ions. With such a stepwise strategy, the self-sorting of individual building blocks was prevented from forming the undesired assemblies, e.g., small macrocycles and coordination polymers. Furthermore, the intra- and intermolecular dynamic exchange study of two supersnowflakes by NMR and mass spectrometry revealed the remarkable stability of these giant supramolecular complexes.

SummaryThe RNA‐guided Cas9 system is a versatile tool for genome editing. Here, we established a RNA‐guided endonuclease (RGEN) system as an in vivo desired‐target mutator (DTM) in maize to reduce the linkage drag during breeding procedure, using the LIGULELESS1 (LG1) locus as a proof‐of‐concept. Our system showed 51.5%–91.2% mutation frequency in T0 transgenic plants. We then crossed the T1 plants stably expressing DTM with six diverse recipient maize lines and found that 11.79%–28.71% of the plants tested were mutants induced by the DTM effect. Analysis of successive F2 plants indicated that the mutations induced by the DTM effect were largely heritable. Moreover, DTM‐generated hybrids had significantly smaller leaf angles that were reduced more than 50% when compared with that of the wild type. Planting experiments showed that DTM‐generated maize plants can be grown with significantly higher density and hence greater yield potential. Our work demonstrate that stably expressed RGEN could be implemented as an in vivo DTM to rapidly generate and spread desired mutations in maize through hybridization and subsequent backcrossing, and hence bypassing the linkage drag effect in convention introgression methodology. This proof‐of‐concept experiment can be a potentially much more efficient breeding strategy in crops employing the RNA‐guided Cas9 genome editing.

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The structures of two dinuclear Cu(II) complexes of dtpb were determined. They are shown to be capable of inducing DNA condensation into nanometer- and micrometer-scale particles under neutral conditions.