Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species.

Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and 'cleaner' knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.

Identification and genetic mapping for rht-DM, a dominant dwarfing gene in mutant semi-dwarf maize using QTL-seq approach.

Semi-dwarfism is an agronomically important trait in breeding for stable high yields and for resistance to damage by wind and rain (lodging resistance). Many QTLs and genes causing dwarf phenotype have been found in maize. However, because of the yield loss associated with these QTLs and genes, they have been difficult to use in breeding for dwarf stature in maize. Therefore, it is important to find the new dwarfing genes or materials without undesirable characters. The objectives of this study were: (1) to figure out the inheritance of semi-dwarfism in mutants; (2) mapping dwarfing gene or QTL. Maize inbred lines '18599' and 'DM173', which is the dwarf mutant derived from the maize inbred line '173' through 60Co-γ ray irradiation. F2 and BC1F1 population were used for genetic analysis. Whole genome resequencing-based technology (QTL-seq) were performed to map dwarfing gene and figured out the SNP markers in predicted region using dwarf bulk and tall bulk from F2 population. Based on the polymorphic SNP markers from QTL-seq, we were fine-mapping the dwarfing gene using F2 population. In F2 population, 398 were dwarf plants and 135 were tall plants. Results of χ2 tests indicated that the ratio of dwarf plants to tall plants was fitted to 3:1 ratio. Furthermore, the χ2 tests of BC1F1 population showed that the ratio was fitted to 1:1 ratio. Based on QTL-seq, the dwarfing gene was located at the region from 111.07 to 124.56 Mb of chromosome 9, and we named it rht-DM. Using traditional QTL mapping with SNP markers, the rht-DM was narrowed down to 400 kb region between SNP-21 and SNP-24. The two SNPs were located at 0.43 and 0.11 cM. Segregation analysis of F2 and BC1F1 indicated that the dwarfing gene was likely a dominant gene. This dwarfing gene was located in the region between 115.02 and 115.42 Mb on chromosome 9.

Identification, Mapping, and Molecular Marker Development for Rgsr8.1: A New Quantitative Trait Locus Conferring Resistance to Gibberella Stalk Rot in Maize (Zea mays L.).

Maize stalk rot is a major fungal disease worldwide, and is difficult to control by chemical methods. Therefore, in maize breeding, quantitative trait loci (QTLs) conferring resistance are important for controlling the disease. Next-generation sequencing technologies are considered a rapid and efficient method to establish the association of agronomic traits with molecular markers or candidate genes. In the present study, we employed QTL-seq, which is a whole-genome resequencing-based approach, to identify candidate genomic regions conferring resistance to maize stalk rot. A novel resistance QTL Rgsr8.1 was finely mapped, conferring broad-spectrum resistance to Gibberella stalk rot (GSR). Segregation analysis in F2 and BC1F1 populations, which were derived from a cross between 18327 (Susceptible) and S72356 (Resistant), indicated that the resistance to GSR was likely to be a quantitatively inherited trait in maize. The result of QTL-seq showed that the resistance to GSR was mapped on chromosome 8 from 161.001 to 170.6 Mb. Based on the simple sequence repeat (SSR) markers, single-nucleotide polymorphism (SNP) markers, and the recombinant test, the location of Rgsr8.1 was narrowed down to 2.04 Mb, flanked by SSR-65 and SNP-25 markers at the physical location from 164.69 to 166.72 Mb based on the maize reference genome. In this region, two candidate resistant genes were found with, one auxin-responsive elements and the other encoding a disease resistance protein. In summary, these results will be useful in maize breeding programs to improve the resistance to GSR in maize.

HIV-TAT-fused FHIT protein functions as a potential pro-apoptotic molecule in hepatocellular carcinoma cells.

Accumulating evidence has demonstrated that FHIT (fragile histidine triad) is a bona fide tumour suppressor gene in a large fraction of human tumours, including hepatocellular cancer. A virus-based delivery system has been developed to transfer the FHIT gene into many types of cancer cells to inhibit growth or even induce apoptosis. However, a protein-based replacement strategy for FHIT has not been performed in cancer cells. Here, we used HIV-TAT (transactivator of transcription)-derived peptide to transfer the purified FHIT protein into HCC (hepatocellular carcinoma) cells and determine the biological effect of this fusion protein in inducing apoptosis. Affinity chromatography was used to purify TAT peptide-fused human FHIT (TAT-FHIT) protein from BL21 Escherichia coli. Immunofluorescence staining and Western blot analysis were performed to identify the expression and internalization of TAT-FHIT in HCC cells compared with the purified FHIT protein. Our study showed that TAT-FHIT protein can translocate into cancer cells in 1 h after incubation at 37°C. Furthermore, the results of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, Annexin-V staining and Western blotting demonstrated that TAT-FHIT can robustly inhibit growth and induce apoptosis of HCC cells in vitro. In addition, a mechanistic study showed that both exogenous and intrinsic apoptotic pathways were involved in TAT-FHIT-mediated apoptosis and this effect could be attenuated partially by a mitochondrial protector TAT-BH4, indicating that mitochondrion plays a critical role in TAT-FHIT-mediated pro-apoptotic effect in cancer cells. Taken together, our study suggests that TAT-FHIT is a potential pro-apoptotic molecule in HCC cells and strengthen the hypothesis of its therapeutic application against HCC.

[Expression and functional analysis of transcriptional factor JunB in hepatic cancer cells].

AIM: To construct the eukaryotic expression vector harboring transcriptional factor JunB and to observe the expression, localization and biological function of JunB in hepatic cancer cells. METHODS: The JunB gene was amplified by PCR from the human liver tissue cDNA library. After confirmed by DNA sequencing in the T-vector, the JunB gene was subcloned into pcDNA3.1(-) and pEGFP-C3 vectors, respectively. The recombinant vectors pcDNA-JunB and pEGFP-JunB were confirmed by Kpn I and BamH I restriction enzyme digestion. The recombinant vectors were transiently transfected into the hepatic cancer cells HepG2 using Lipofectamin2000. Western blotting was used to detect the expression of exogenous JunB and fluorescence microscopy was applied to observe the localization of JunB protein coupled with enhanced green fluorescent protein (EGFP) in hepatic cancer cells. Double luciferase reporter assay was performed to determine the effect of JunB on the transcriptional regulation of target genes. RESULTS: The recombinant eukaryotic expression vector carrying JunB or JunB-EGFP fusion gene was constructed and transiently transfected into HepG2 cells. Western blot analysis confirmed the exogenous expression of JunB. JunB-EGFP was observed uniquely in the nuclei. Double luciferase assay showed the transcriptional up-regulation of the VEGF in the presence of JunB. CONCLUSION: We constructed the recombinant eukaryotic expression vectors harboring JunB and JunB-EGFP successfully. The exogenous JunB is localized in the nuclei of transiently transfected HepG2 cells, suggesting JunB may function as a transcriptional factor. The result of reporter assay showed that JunB up-regulates the expression of VEGF, a tumor-angiogenesis associated molecule, at a transcriptional level. These demonstrate that JunB might be a novel target for an anti-angiogenesis treatment for hepatic cancers.

Significant improvement of stress tolerance in tobacco plants by overexpressing a stress-responsive aldehyde dehydrogenase gene from maize (Zea mays).

Aldehyde dehydrogenases (ALDHs) play a central role in detoxification processes of aldehydes generated in plants when exposed to the stressed conditions. In order to identify genes required for the stresses responses in the grass crop Zea mays, an ALDH (ZmALDH22A1) gene was isolated and characterized. ZmALDH22A1 belongs to the family ALDH22 that is currently known only in plants. The ZmALDH22A1 encodes a protein of 593 amino acids that shares high identity with the orthologs from Saccharum officinarum (95%), Oryza sativa (89%), Triticum aestivum (87%) and Arabidopsis thaliana (77%), respectively. Real-time PCR analysis indicates that ZmALDH22A1 is expressed differentially in different tissues. Various elevated levels of ZmALDH22A1 expression have been detected when the seedling roots exposed to abiotic stresses including dehydration, high salinity and abscisic acid (ABA). Tomato stable transformation of construct expressing the ZmALDH22A1 signal peptide fused with yellow fluorescent protein (YFP) driven by the CaMV35S-promoter reveals that the fusion protein is targeted to plastid. Transgenic tobacco plants overexpressing ZmALDH22A1 shows elevated stresses tolerance. Stresses tolerance in transgenic plants is accompanied by a reduction of malondialdehyde (MDA) derived from cellular lipid peroxidation.

Functional defect at the rice choline monooxygenase locus from an unusual post-transcriptional processing is associated with the sequence elements of short-direct repeats.

Glycine betaine (GB), a quaternary ammonium solute, plays a crucial role in developing osmotic tolerance. Rice contains a choline monooxygenase (CMO) and two betaine aldehyde dehydrogenase homologues that are required for GB synthesis, but usually no GB is accumulated in rice (Oryza sativa). To elucidate the molecular processes that underlie the GB deficiency in rice, an experiment involving rice and spinach (Spinacia oleracea) was conducted to analyze the products transcribed from CMO genes. Reverse transcription-polymerase chain reaction (RT-PCR) was used to obtain CMO transcripts and a sequencing approach was employed to analyze the structural composition of various CMO transcripts. The results showed that most rice CMO transcripts were processed incorrectly, retaining introns or deleted of coding sequences; the unusual deletion events occurred at sequence elements of the short-direct repeats. In conclusion, the production of incorrect CMO transcripts results in a deficiency of the full-length CMO protein and probably reduces GB accumulation considerably in rice plants. Sequence comparison results also implied that the unusual deletion-site selection might be mediated by the short-direct repeats in response to stress conditions.