ABSTRACT
Leaf senescence is a vital aspect of plant physiology and stress responses and is induced by endogenous factors and environmental cues. The plant-specific NAC (NAM, ATAF1/2, CUC2) transcription factor family influences growth, development, and stress responses in Arabidopsis (Arabidopsis thaliana) and other species. However, the roles of NACs in tobacco (Nicotiana tabacum) leaf senescence are still unclear. Here, we report that NtNAC56 regulates leaf senescence in tobacco. Transgenic plants overexpressing NtNAC56 (NtNAC56-OE) showed induction of senescence-related genes and exhibited early senescence and lower chlorophyll content compared to wild-type (WT) plants and the Ntnac56-19 mutant. In addition, root development and seed germination were inhibited in the NtNAC56-OE lines. Transmission electron microscopy observations accompanied by physiological and biochemical assays revealed that NtNAC56 overexpression triggers chloroplast degradation and reactive oxygen species accumulation in tobacco leaves. Transcriptome analysis demonstrated that NtNAC56 activates leaf senescence-related genes and jasmonic acid (JA) biosynthesis pathway genes. In addition, the JA content of NtNAC56-OE plants was higher than in WT plants, and JA treatment induced NtNAC56 expression. We performed DNA affinity purification sequencing to identify direct targets of NtNAC56, among which we focused on LIPOXYGENASE 5 (NtLOX5), a key gene in JA biosynthesis. A dual-luciferase reporter assay and a yeast one-hybrid assay confirmed that NtNAC56 directly binds to the TTTCTT motif in the NtLOX5 promoter. Our results reveal a mechanism whereby NtNAC56 regulates JA-induced leaf senescence in tobacco and provide a strategy for genetically manipulating leaf senescence and plant growth.
Subject(s)
Cyclopentanes , Gene Expression Regulation, Plant , Nicotiana , Oxylipins , Plant Leaves , Plant Proteins , Plant Senescence , Plants, Genetically Modified , Transcription Factors , Nicotiana/genetics , Nicotiana/physiology , Nicotiana/drug effects , Nicotiana/growth & development , Oxylipins/metabolism , Oxylipins/pharmacology , Cyclopentanes/metabolism , Cyclopentanes/pharmacology , Plant Leaves/metabolism , Plant Leaves/genetics , Plant Leaves/physiology , Plant Senescence/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Plant Proteins/metabolism , Plant Proteins/genetics , Reactive Oxygen Species/metabolism , Chlorophyll/metabolism , Chloroplasts/metabolism , Chloroplasts/ultrastructure , Promoter Regions, Genetic/geneticsABSTRACT
KEY MESSAGE: We developed an efficient promoter editing method to create different weak Ehd1 alleles in elite japonica rice variety ZJ8 with slightly delayed heading and improved yield for use in breeding. Heading date is an important agronomic trait of rice (Oryza sativa) that determines the planting areas and cultivation seasons of different varieties, thus affecting final yield. Early heading date 1 (Ehd1) is a major rice integrator gene in the regulatory network of heading date whose expression level is negatively correlated with heading date and grain yield. Some elite japonica varieties such as Zhongjia 8 (ZJ8) show very early heading with poor agronomic traits when planted in South China. This problem can be addressed by downregulating the expression of Ehd1. In this study, we analyzed the cis-regulatory elements in the Ehd1 promoter region. We then used CRISPR/Cas9-mediated editing to modify the Ehd1 promoter at multiple target sites in ZJ8. We rapidly identified homozygous allelic mutations in the T2 generation via long-read sequencing. We obtained several Ehd1 promoter mutants with different degrees of lower Ehd1 expression, delayed heading date, and improved yield-related traits. We developed an efficient promoter editing method to create different weak Ehd1 alleles for breeding selection. Using this method, a series of heading date materials from elite varieties can be created to expand the planting area of rice and improve grain yields.
Subject(s)
Oryza , Oryza/genetics , Plant Breeding , Promoter Regions, Genetic , Agriculture , Alleles , Edible Grain/geneticsABSTRACT
KEY MESSAGE: We clarify the influence of the genotypes of the heading date genes Hd1, Ghd7, DTH8, and PRR37 and their combinations on yield-related traits and the functional differences between different haplotypes. Heading date is a key agronomic trait in rice (Oryza sativa L.) that determines yield and adaptability to different latitudes. Heading date 1 (Hd1), Grain number, plant height, and heading date 7 (Ghd7), Days to heading on chromosome 8 (DTH8), and PSEUDO-RESPONSE REGULATOR 37 (PRR37) are core rice genes controlling photoperiod sensitivity, and these genes have many haplotypes in rice cultivars. However, the effects of different haplotypes at these genes on yield-related traits in diverse rice materials remain poorly characterized. In this study, we knocked out Hd1, Ghd7, DTH8, or PRR37, alone or together, in indica and japonica varieties and systematically investigated the agronomic traits of each knockout line. Ghd7 and PRR37 increased the number of spikelets and improved yield, and this effect was enhanced with the Ghd7 DTH8 or Ghd7 PRR37 combination, but Hd1 negatively affected yield. We also identified a new weak functional Ghd7 allele containing a mutation that interferes with splicing. Furthermore, we determined that the promotion or inhibition of heading date by different PRR37 haplotypes is related to PRR37 expression levels, day length, and the genetic background. For rice breeding, a combination of functional alleles of Ghd7 and DTH8 or Ghd7 and PRR37 in the hd1 background can be used to increase yield. Our study clarifies the effects of heading date genes on yield-related traits and the functional differences among their different haplotypes, providing valuable information to identify and exploit elite haplotypes for heading date genes to breed high-yielding rice varieties.
Subject(s)
Oryza , Oryza/metabolism , Plant Breeding , Phenotype , Mutation , Genotype , Plant Proteins/genetics , Plant Proteins/metabolism , Gene Expression Regulation, Plant , Flowers/genetics , PhotoperiodABSTRACT
Rapeseed (Brassica napus) is a major edible oilseed crop consumed worldwide. However, its yield is seriously affected by infection from the broad-spectrum non-obligate pathogen Sclerotinia sclerotiorum due to a lack of highly resistant germplasm. Here, we identified a Sclerotinia-resistant and light-dependent lesion mimic mutant from an ethyl methanesulfonate-mutagenized population of the rapeseed inbred Zhongshuang 11 (ZS11) named lesion mimic mutant 1 (lmm1). The phenotype of lmm1 is controlled by a single recessive gene, named LESION MIMIC MUTANT 1 (LMM1), which mapped onto chromosome C04 by bulked segregant analysis within a 2.71-Mb interval. Histochemical analysis indicated that H2O2 strongly accumulated and cell death occurred around the lesion mimic spots. Among 877 differentially expressed genes (DEGs) between ZS11 and lmm1 leaves, 188 DEGs were enriched in the defense response, including 95 DEGs involved in systemic acquired resistance, which is consistent with the higher salicylic acid levels in lmm1. Combining bulked segregant analysis and transcriptome analysis, we identified a significantly up-regulated gene, BnaC4.PR2, which encodes ß-1,3-glucanase, as the candidate gene for LMM1. Overexpression of BnaC4.PR2 may induce a reactive oxygen species burst to trigger partial cell death and systemic acquired resistance. Our study provides a new genetic resource for S. sclerotiorum resistance as well as new insights into disease resistance breeding in B. napus.
Subject(s)
Ascomycota , Brassica napus , Brassica rapa , Brassica napus/genetics , Brassica napus/metabolism , Hydrogen Peroxide/metabolism , Plant Diseases/genetics , Plant Breeding , Brassica rapa/genetics , Ascomycota/physiology , Disease Resistance/geneticsABSTRACT
BACKGROUND: The pod shattering (PS) trait negatively affects the crop yield in rapeseed especially under dry conditions. To better understand the trait and cultivate higher resistance varieties, it's necessary to identify key genes and unravel the PS mechanism thoroughly. RESULTS: In this study, we conducted a comparative transcriptome analysis between two materials significantly different in silique shatter resistance lignin deposition and polygalacturonase (PG) activity. Here, we identified 10,973 differentially expressed genes at six pod developmental stages. We found that the late pod development stages might be crucial in preparing the pods for upcoming shattering events. GO enrichment results from K-means clustering and weighed gene correlation network analysis (WGCNA) both revealed senescence-associated genes play an important role in PS. Two hub genes Bna.A05ABI5 and Bna.C03ERF/AP2-3 were selected from the MEyellow module, which possibly regulate the PS through senescence-related mechanisms. Further investigation found that senescence-associated transcription factor Bna.A05ABI5 upregulated the expression of SAG2 and ERF/AP2 to control the shattering process. In addition, the upregulation of Bna.C03ERF/AP2-3 is possibly involved in the transcription of downstream SHP1/2 and LEA proteins to trigger the shattering mechanism. We also analyzed the PS marker genes and found Bna.C07SHP1/2 and Bna.PG1/2 were significantly upregulated in susceptible accession. Furthermore, the role of auxin transport by Bna.WAG2 was also observed, which could reduce the PG activity to enhance the PS resistance through the cell wall loosening process. CONCLUSION: Based on comparative transcriptome evaluation, this study delivers insights into the regulatory mechanism primarily underlying the variation of PS in rapeseed. Taken together, these results provide a better understanding to increase the yield of rapeseed by reducing the PS through better engineered crops.
ABSTRACT
CRISPR/Cas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) can efficiently mediate C-to-T/G-to-A and A-to-G/T-to-C substitutions, respectively; however, achieving base transversions (C-to-G/C-to-A and A-to-T/A-to-C) is challenging and has been rarely studied in plants. Here, we constructed new plant C-to-G base editors (CGBEs) and new A-to-Y (T/C) base editors and explored their base editing characteristics in rice. First, we fused the highly active cytidine deaminase evoFENRY and the PAM-relaxed Cas9-nickase variant Cas9n-NG with rice and human uracil DNA N-glycosylase (rUNG and hUNG), respectively, to construct CGBE-rUNG and CGBE-hUNG vector tools. The analysis of five NG-PAM target sites showed that these CGBEs achieved C-to-G conversions with monoallelic editing efficiencies of up to 27.3% in T0 rice, with major byproducts being insertion/deletion mutations. Moreover, for the A-to-Y (C or T) editing test, we fused the highly active adenosine deaminase TadA8e and the Cas9-nickase variant SpGn (with NG-PAM) with Escherichia coli endonuclease V (EndoV) and human alkyladenine DNA glycosylase (hAAG), respectively, to generate ABE8e-EndoV and ABE8e-hAAG vectors. An assessment of five NG-PAM target sites showed that these two vectors could efficiently produce A-to-G substitutions in a narrow editing window; however, no A-to-Y editing was detected. Interestingly, the ABE8e-EndoV also generated precise small fragment deletions in the editing window from the 5'-deaminated A base to the SpGn cleavage site, suggesting its potential value in producing predictable small-fragment deletion mutations. Overall, we objectively evaluated the editing performance of CGBEs in rice, explored the possibility of A-to-Y editing, and developed a new ABE8e-EndoV tool, thus providing a valuable reference for improving and enriching base editing tools in plants.
Subject(s)
Gene Editing , Oryza , CRISPR-Cas Systems/genetics , Deoxyribonuclease I/genetics , Escherichia coli/genetics , Guanine/analogs & derivatives , Humans , Oryza/geneticsABSTRACT
Heading date determines the seasonal and regional adaptation of rice (Oryza sativa L.) varieties and is mainly controlled by photoperiod sensitivity (PS). The core heading date genes Hd1, Ghd7, DTH8, and PRR37 act synergistically in regulating the PS. In this study, we systematically analyze the heading date, PS, and agronomic traits of eight homozygous lines with various combinations of Hd1, Ghd7, and DTH8 alleles in the prr37 background under long-day (LD) and short-day (SD) conditions, respectively. We find that Hd1 alone promotes heading, regardless of the day length. However, under LDs, Hd1 suppresses flowering, in coordination with functional Ghd7 or with Ghd7 and DTH8. These loci cooperate to negatively regulate the Ehd1-Hd3a/RFT1 pathway and delay heading. Under SDs, Hd1 competes with various heading suppressors to promote heading. Therefore, the dual function of Hd1 is vital for PS. The lines carrying Hd1 alone show reduced plant height with fewer primary and secondary branches in panicles. Lines carrying Ghd7 and DTH8 (with hd1) show delayed heading and improve agronomic traits. Overall, our results reveal the regulation of rice PS flowering by the core heading date genes and their effects on agronomic traits, providing valuable information for the selection of rice varieties for adaptation to different light and temperature conditions.
Subject(s)
Oryza , Flowers/metabolism , Gene Expression Regulation, Plant/genetics , Oryza/metabolism , Photoperiod , Plant Proteins/genetics , Plant Proteins/metabolismABSTRACT
BACKGROUND: In the oilseed crop Brassica napus (rapeseed), various metabolic processes influence seed oil content, oil quality, and biological yield. However, the role of plastid membrane proteins in these traits has not been explored. RESULTS: Our genome-wide association study (GWAS) of 520 B. napus accessions identified the chloroplast membrane protein-localized FATTY ACID EXPORTER 1-1 (FAX1-1) as a candidate associated with biological yield. Seed transcript levels of BnaFAX1-1 were higher in a cultivar with high seed oil content relative to a low-oil cultivar. BnaFAX1-1 was localized to the plastid envelope. When expressed in Arabidopsis thaliana, BnaFAX1-1 enhanced biological yield (total plant dry matter), seed yield and seed oil content per plant. Likewise, in the field, B. napus BnaFAX1-1 overexpression lines (BnaFAX1-1-OE) displayed significantly enhanced biological yield, seed yield, and seed oil content compared with the wild type. BnaFAX1-1 overexpression also up-regulated gibberellic acid 4 (GA4) biosynthesis, which may contribute to biological yield improvement. Furthermore, oleic acid (C18:1) significantly increased in BnaFAX1-1 overexpression seeds. CONCLUSION: Our results indicated that the putative fatty acid exporter BnaFAX1-1 may simultaneously improve seed oil content, oil quality and biological yield in B. napus, providing new approaches for future molecular breeding.
ABSTRACT
BACKGROUND: Noninvasive prenatal testing (NIPT) is one of the most commonly employed clinical measures for screening of fetal aneuploidy. Fetal Fraction (ff) has been demonstrated to be one of the key factors affecting the performance of NIPT. Accurate quantification of ff plays vital role in NIPT. METHODS: In this study, we present a new approach, the accurate Quantification of Fetal Fraction with Shallow-Coverage sequencing of maternal plasma DNA (FF-QuantSC), for the estimation of ff in NIPT. The method employs neural network model and utilizes differential genomic patterns between fetal and maternal genomes to quantify ff. RESULTS: Our results show that the predicted ff by FF-QuantSC exhibit high correlation with the Y chromosome-based method on male pregnancies, and achieves the highest accuracy compared with other ff estimation approaches. We also demonstrate that the model generates statistically similar results on both male and female pregnancies. CONCLUSION: FF-QuantSC achieves high accuracy in ff quantification. The method is suitable for application in both male and female pregnancies. Since the method does not require additional information upon NIPT routines, it can be easily incorporated into current NIPT settings without causing extra costs. We believe that FF-QuantSC shall provide valuable additions to NIPT.
Subject(s)
Neural Networks, Computer , Noninvasive Prenatal Testing/methods , Sequence Analysis, DNA/methods , Adult , Female , Humans , Noninvasive Prenatal Testing/standards , Pregnancy , Sensitivity and Specificity , Sequence Analysis, DNA/standards , SoftwareABSTRACT
Triacylglycerols (TAGs) are the major storage form of seed oil in oilseed plants. They are biosynthesized de novo in seed plastids and then transported into the endoplasmic reticulum. However, the transport mechanism for plastid fatty acids in developing seeds remains unknown. Here, we isolated two novel plastid fatty acid exporters (FATTYACID EXPORT 2 [FAX2] and FAX4, respectively) specifically abundant in seed embryos during the seed-filling stage in Arabidopsis (Arabidopsis thaliana). FAX2 and FAX4 were both localized to the chloroplast membrane. FAX2 and FAX4 loss-of-function mutations caused deficiencies in embryo and cotyledon development. Seeds of fax2fax4 double mutants exhibited significantly reduced TAG contents but elevated levels of plastid lipid contents compared with those of wild-type plants. By contrast, overexpression of FAX2 or FAX4 enhanced TAG deposition. Seed-feeding experiments showed that the two FAX proteins transported 14C-plastid fatty acids and 13C-oleic acids for TAG biosynthesis during the seed-filling stage. Together, our data demonstrate that FAX2 and FAX4 play critical roles in transporting plastid fatty acids for TAG biosynthesis during seed embryo development. These two transporters may have broad application for increasing oil yield in oilseed crops.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Fatty Acids/metabolism , Plant Oils/metabolism , Seeds/metabolism , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Triglycerides/metabolismABSTRACT
Cotton (Gossypium hirsutum L.) is a major crop and the main source of natural fiber worldwide. Because various abiotic and biotic stresses strongly influence cotton fiber yield and quality, improved stress resistance of this crop plant is urgently needed. In this study, we used Gateway technology to construct a normalized full-length cDNA overexpressing (FOX) library from upland cotton cultivar ZM12 under various stress conditions. The library was transformed into Arabidopsis to produce a cotton-FOX-Arabidopsis library. Screening of this library yielded 6,830 transgenic Arabidopsis lines, of which 757 were selected for sequencing to ultimately obtain 659 cotton ESTs. GO and KEGG analyses mapped most of the cotton ESTs to plant biological process, cellular component, and molecular function categories. Next, 156 potential stress-responsive cotton genes were identified from the cotton-FOX-Arabidopsis library under drought, salt, ABA, and other stress conditions. Four stress-related genes identified from the library, designated as GhCAS, GhAPX, GhSDH, and GhPOD, were cloned from cotton complementary DNA, and their expression patterns under stress were analyzed. Phenotypic experiments indicated that overexpression of these cotton genes in Arabidopsis affected the response to abiotic stress. The method developed in this study lays a foundation for high-throughput cloning and rapid identification of cotton functional genes.
Subject(s)
Arabidopsis/genetics , DNA, Complementary/genetics , Gene Library , Genes, Plant , Gossypium/genetics , Stress, Physiological/genetics , Abscisic Acid/pharmacology , Arabidopsis/growth & development , Droughts , Gene Expression Regulation, Plant/drug effects , Phenotype , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/drug effects , Plant Roots/genetics , Plant Roots/growth & development , Plants, Genetically Modified , Seedlings/drug effects , Seeds/drug effects , Seeds/genetics , Subcellular Fractions/metabolismABSTRACT
BACKGROUND: Increasing seed oil content is one of the most important targets for rapeseed (Brassica napus) breeding. However, genetic mechanisms of mature seed oil content in Brassica napus (B. napus) remain little known. To identify oil content-related genes, a genome-wide association study (GWAS) was performed using 588 accessions. RESULTS: High-throughput genome resequencing resulted in 385,692 high-quality single nucleotide polymorphism (SNPs) with a minor allele frequency (MAF) > 0.05. We identified 17 loci that were significantly associated with seed oil content, among which 12 SNPs were distributed on the A3 (11 loci) and A1 (one loci) chromosomes, and five novel significant SNPs on the C5 (one loci) and C7 (four loci) chromosomes, respectively. Subsequently, we characterized differentially expressed genes (DEGs) between the seeds and silique pericarps on main florescences and primary branches of extremely high- and low-oil content accessions (HO and LO). A total of 64 lipid metabolism-related DEGs were identified, 14 of which are involved in triacylglycerols (TAGs) biosynthesis and assembly. Additionally, we analyzed differences in transcription levels of key genes involved in de novo fatty acid biosynthesis in the plastid, TAGs assembly and lipid droplet packaging in the endoplasmic reticulum (ER) between high- and low-oil content B. napus accessions. CONCLUSIONS: The combination of GWAS and transcriptome analyses revealed seven candidate genes located within the confidence intervals of significant SNPs. Current findings provide valuable information for facilitating marker-based breeding for higher seed oil content in B. napus.
ABSTRACT
The study of Mendelian diseases and the identification of their causative genes are of great significance in the field of genetics. The evaluation of the pathogenicity of genes and the total number of Mendelian disease genes are both important questions worth studying. However, very few studies have addressed these issues to date, so we attempt to answer them in this study. We calculated the gene pathogenicity prediction (GPP) score by a machine learning approach (random forest algorithm) to evaluate the pathogenicity of genes. When we applied the GPP score to the testing gene set, we obtained an accuracy of 80%, recall of 93% and area under the curve of 0.87. Our results estimated that a total of 10,384 protein-coding genes were Mendelian disease genes. Furthermore, we found the GPP score was positively correlated with the severity of disease. Our results indicate that GPP score may provide a robust and reliable guideline to predict the pathogenicity of protein-coding genes. To our knowledge, this is the first trial to estimate the total number of Mendelian disease genes.
Subject(s)
Algorithms , Genetic Diseases, Inborn/genetics , Genetic Predisposition to Disease/genetics , Machine Learning , Genes, Dominant/genetics , Genes, Recessive/genetics , Genetic Diseases, Inborn/diagnosis , Humans , ROC CurveABSTRACT
BACKGROUND: In the past few decades, microalgae biofuel has become one of the most interesting sources of renewable energy. However, the higher cost of microalgae biofuel compared to that of petroleum prevented microalgae biofuel production. Therefore, the research on increasing lipid productivity from microalgae becomes more important. The lipid production source, triacylglycerol biosynthesis in microalgae requires short chain fatty acids as substrates, which are synthesized in chloroplasts. However, the transport mechanism of fatty acids from microalgae chloroplasts to cytosol remains unknown. RESULTS: cDNAs from two homologs of the Arabidopsis fatty acid exporter 1 (FAX1) were cloned from Chlamydomonas reinhardtii and were named crfax1 and crfax2. Both CrFAXs were involved in fatty acid transport, and their substrates were mainly C16 and C18 fatty acids. Overexpression of both CrFAXs increased the accumulation of the total lipid content in algae cells, and the fatty acid compositions were changed under normal TAP or nitrogen deprivation conditions. Overexpression of both CrFAXs also increased the chlorophyll content. The MGDG content was decreased but the TAG, DAG, DGDG and other lipid contents were increased in CrFAXs overexpression strains. CONCLUSION: These results reveal that CrFAX1 and CrFAX2 were involved in mediating fatty acid export for lipids biosynthesis in C. reinhardtii. In addition, overexpression of both CrFAXs obviously increased the intracellular lipid content, especially the triacylglycerol content in microalgae, which provides a potential technology for the production of more biofuels using microalgae.
ABSTRACT
WEGO (Web Gene Ontology Annotation Plot), created in 2006, is a simple but useful tool for visualizing, comparing and plotting GO (Gene Ontology) annotation results. Owing largely to the rapid development of high-throughput sequencing and the increasing acceptance of GO, WEGO has benefitted from outstanding performance regarding the number of users and citations in recent years, which motivated us to update to version 2.0. WEGO uses the GO annotation results as input. Based on GO's standardized DAG (Directed Acyclic Graph) structured vocabulary system, the number of genes corresponding to each GO ID is calculated and shown in a graphical format. WEGO 2.0 updates have targeted four aspects, aiming to provide a more efficient and up-to-date approach for comparative genomic analyses. First, the number of input files, previously limited to three, is now unlimited, allowing WEGO to analyze multiple datasets. Also added in this version are the reference datasets of nine model species that can be adopted as baselines in genomic comparative analyses. Furthermore, in the analyzing processes each Chi-square test is carried out for multiple datasets instead of every two samples. At last, WEGO 2.0 provides an additional output graph along with the traditional WEGO histogram, displaying the sorted P-values of GO terms and indicating their significant differences. At the same time, WEGO 2.0 features an entirely new user interface. WEGO is available for free at http://wego.genomics.org.cn.
Subject(s)
Gene Ontology , Internet , Molecular Sequence Annotation/methods , Software , Databases, Genetic , High-Throughput Nucleotide Sequencing , User-Computer InterfaceABSTRACT
Background: Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. Findings: We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5Î untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription. Conclusion: Our results prove that dCas9 methyltransferases cause efficient RNA-guided methylation of specific endogenous CpGs. However, there is significant off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are required.
Subject(s)
DNA Methylation/genetics , Genome/genetics , RNA, Guide, Kinetoplastida/genetics , CRISPR-Cas Systems/genetics , CpG Islands/genetics , HEK293 Cells , Humans , Methyltransferases/geneticsABSTRACT
Hundreds of thousands of human genomes are now being sequenced to characterize genetic variation and use this information to augment association mapping studies of complex disorders and other phenotypic traits. Genetic variation is identified mainly by mapping short reads to the reference genome or by performing local assembly. However, these approaches are biased against discovery of structural variants and variation in the more complex parts of the genome. Hence, large-scale de novo assembly is needed. Here we show that it is possible to construct excellent de novo assemblies from high-coverage sequencing with mate-pair libraries extending up to 20 kilobases. We report de novo assemblies of 150 individuals (50 trios) from the GenomeDenmark project. The quality of these assemblies is similar to those obtained using the more expensive long-read technology. We use the assemblies to identify a rich set of structural variants including many novel insertions and demonstrate how this variant catalogue enables further deciphering of known association mapping signals. We leverage the assemblies to provide 100 completely resolved major histocompatibility complex haplotypes and to resolve major parts of the Y chromosome. Our study provides a regional reference genome that we expect will improve the power of future association mapping studies and hence pave the way for precision medicine initiatives, which now are being launched in many countries including Denmark.
Subject(s)
Genetic Variation/genetics , Genetics, Population/standards , Genome, Human/genetics , Genomics/standards , Sequence Analysis, DNA/standards , Adult , Alleles , Child , Chromosomes, Human, Y/genetics , Denmark , Female , Haplotypes/genetics , Humans , Major Histocompatibility Complex/genetics , Male , Maternal Age , Mutation Rate , Paternal Age , Point Mutation/genetics , Reference StandardsABSTRACT
Derivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to shed more light on the underlying biological mechanisms of porcine pluripotency. LIF-derived piPSCs were more successful than their FGF-derived counterparts in the generation of in vitro chimeras and in teratoma formation. When LIF piPSCs chimeras were transferred into surrogate sows and allowed to develop, only their prescence within the embryonic membranes could be detected. Whole-transcriptome analysis of the piPSCs and porcine neonatal fibroblasts showed that they clustered together, but apart from the two pluripotent cell populations of early porcine embryos, indicating incomplete reprogramming. Indeed, bioinformatic analysis of the pluripotency-related gene network of the LIF- versus FGF-derived piPSCs revealed that ZFP42 (REX1) expression was absent in both piPSC-like cells, whereas it was expressed in the porcine inner cell mass at Day 7/8. A second striking difference was the expression of ATOH1 in piPSC-like cells, which was absent in the inner cell mass. Moreover, our gene expression analyses plus correlation analyses of known pluripotency genes identified unique relationships between pluripotency genes in the inner cell mass, which are to some extent, in the piPSC-like cells. This deficiency in downstream gene activation and divergent gene expression may be underlie the inability to derive germ line-transmitting piPSCs, and provides unique insight into which genes are necessary to achieve fully reprogrammed piPSCs. 84: 229-245, 2017. © 2016 Wiley Periodicals, Inc.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Fibroblast Growth Factors/pharmacology , Gene Expression Regulation/drug effects , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Transcription Factors/metabolism , Leukemia Inhibitory Factor/pharmacology , Animals , Induced Pluripotent Stem Cells/cytology , SwineABSTRACT
Biased integration remains a key challenge for gene therapy based on lentiviral vector technologies. Engineering of next-generation lentiviral vectors targeting safe genomic harbors for insertion is therefore of high relevance. In a previous paper (Cai et al., 2014a), we showed the use of integrase-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR). Here, we follow this strategy to engineer ZFN-loaded IDLVs that insert transgenes by a homology-driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. We show targeted gene integration in human stem cells, including CD34(+) hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded 'all-in-one' IDLVs for site-directed gene insertion in stem cell-based gene therapies.
Subject(s)
Gene Targeting/methods , Genetic Therapy/methods , Genetic Vectors , Homologous Recombination , Lentivirus/genetics , Mutagenesis, Insertional , Stem Cells/physiology , Cells, Cultured , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , Deoxyribonucleases/genetics , Deoxyribonucleases/metabolism , HumansABSTRACT
The primary therapeutic effects of Chinese herbal medicine (CHM) are based on the properties of each herb and the strategic combination of herbs in formulae. The herbal formulae are constructed according to Chinese medicine theory: the "Traditional Principles for Constructing Chinese Herbal Medicinal Formulae" and the "Principles of Combining Medicinal Substances." These principles of formulation detail how and why multiple medicinal herbs with different properties are combined together into a single formula. However, the concept of herbal synergism in CHM still remains a mystery due to lack of scientific data and modern assessment methods. The Compound Danshen Formula (CDF) is a validated formula that has been used to treat a variety of diseases for hundreds of years in China and other countries. The CDF will be employed to illustrate the theory and principle of Chinese herbal medicine formulation. The aim of this review is to describe how Chinese herbal medicinal formulae are constructed according to Chinese medicine theory and to illustrate with scientific evidence how Chinese herbs work synergistically within a formula, thereby supporting Chinese medicine theory and practice.