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2.
Nanomaterials (Basel) ; 10(10)2020 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-32977574

RESUMO

V2O5-WO3/TiO2 as a commercial selective catalytic reduction (SCR) catalyst usually used at middle-high temperatures was modified by loading of MnOx for the purpose of enhancing its performance at lower temperatures. Manganese oxides were loaded onto V-W/Ti monolith by the methods of impregnation (I), precipitation (P), and in-situ growth (S), respectively. SCR activity of each modified catalyst was investigated at temperatures in the range of 100-340 °C. Catalysts were characterized by specific surface area and pore size determination (BET), X-ray diffraction (XRD), temperature programmed reduction (TPR), etc. Results show that the loading of MnOx remarkably enhanced the SCR activity at a temperature lower than 280 °C. The catalyst prepared by the in-situ growth method was found to be most active for SCR.

3.
ACS Nano ; 2020 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-32931252

RESUMO

Implant-related infections (IRIs) are a serious complication after orthopedic surgery, especially when a biofilm develops and establishes physical and chemical barriers protecting bacteria from antibiotics and the hosts local immune system. Effectively eliminating biofilms is essential but difficult, as it requires not only breaking the physical barrier but also changing the chemical barrier that induces an immunosuppressive microenvironment. Herein, tailored to a biofilm microenvironment (BME), we proposed a space-selective chemodynamic therapy (CDT) strategy to combat IRIs using metastable CuFe5O8 nanocubes (NCs) as smart Fenton-like reaction catalysts whose activity can be regulated by pH and H2O2 concentration. In the biofilm, extracellular DNA (eDNA) was cleaved by high levels of hydroxyl radicals (•OH) catalyzed by CuFe5O8 NCs, thereby disrupting the rigid biofilm. Outside the biofilm with relatively higher pH and lower H2O2 concentration, lower levels of generated •OH effectively reversed the immunosuppressive microenvironment by inducing pro-inflammatory macrophage polarization. Biofilm fragments and exposed bacteria were then persistently eliminated through the collaboration of pro-inflammatory immunity and •OH. The spatially selective activation of CDT and synergistic immunomodulation exerted excellent effects on the treatment of IRIs in vitro and in vivo. The anti-infection strategy is expected to provide a method to conquer IRIs.

5.
J Exp Bot ; 71(19): 6128-6141, 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-32640017

RESUMO

High temperature stress is an inevitable environmental factor in certain geographical regions. To study the effect of day and night high temperature stress on male reproduction, the heat-sensitive cotton line H05 was subjected to high temperature stress. High day/normal night (HN) and normal day/high night (NH) temperature treatments were compared with normal day/normal night (NN) temperature as a control. At the anther dehiscence stage, significant differences were observed, with a reduction in flower size and filament length, and sterility in pollen, seen in NH more than in HN. A total of 36 806 differentially expressed genes were screened, which were mainly associated with fatty acid and jasmonic acid (JA) metabolic pathways. Fatty acid and JA contents were reduced more in NH than HN. Under NH, ACYL-COA OXIDASE 2 (ACO2), a JA biosynthesis gene, was down-regulated. Interestingly, aco2 CRISPR-Cas9 mutants showed male sterility under the NN condition. The exogenous application of methyl jasmonate to early-stage buds of mutants rescued the sterile pollen and indehiscent anther phenotypes at the late stage. These data show that high temperature at night may affect fatty acid and JA metabolism in anthers by suppressing GhACO2 and generate male sterility more strongly than high day temperature.

6.
Food Chem ; 332: 127431, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32645668

RESUMO

Illegal usage of ß-agonists as the animal growth promoters can lead to multiple harmful impacts to public health, thus detection of ß-agonists at trace level in complex sample matrixes is of great importance. In recent years, emergence of advanced nanomaterials greatly facilitates the advancement of sensors in terms of sensitivity, specificity and robustness. Plenty of nanoparticles-based sensors have been developed for ß-agonists determination. In this review, we comprehensively summarized the construction of emerging nanoparticles-based sensors (including colorimetric sensors, fluorescent sensors, chemiluminescent sensors, electrochemical sensors, electrochemiluminescent sensors, surface enhanced Raman scattering sensors, surface plasmon resonance sensors, quartz crystal microbalance sensors, etc.), and nanomaterial-based enzyme-linked immunosorbent assay (nano-ELISA). Impressively, the applications of nanoparticles-based sensors and nano-ELISAs in the detection of ß-agonists have also been summarized and discussed. In the end, future opportunities and challenges in the design construction of nanoparticles (NPs)-based sensors and their applications in ß-agonist assay are tentatively proposed.


Assuntos
Agonistas Adrenérgicos/análise , Nanoestruturas/química , Animais , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Colorimetria/instrumentação , Colorimetria/métodos , Humanos , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodos , Ressonância de Plasmônio de Superfície/instrumentação , Ressonância de Plasmônio de Superfície/métodos
7.
Connect Tissue Res ; : 1-10, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32500755

RESUMO

AIMS: Inflammatory responses to wear debris cause osteolysis that leads to aseptic loosening and hip arthroplasty failure. Wear debris stimulate macrophages and fibroblasts to secret proinflammatory cytokines, including TNF-α and IL-6, which have been specifically implicated in periprosthetic osteolysis and osteoclast differentiation. Naringin has anti-inflammatory effect in macrophages. Moreover, naringin inhibited osteoclastogenesis and wear particles-induced osteolysis. In this study, we examined the potential inhibitory effects of naringin on titanium (Ti) particle-induced proinflammatory cytokines secretion in fibroblasts and the possible underlying molecular mechanisms. MATERIALS AND METHODS: Fibroblasts were isolated from periprosthetic membrane at the time of revision surgery performed due to aseptic loosening after hip arthroplasty and were cultured in the presence or absence of Ti particles, naringin and mitogen-activated protein kinase (MAPK) inhibitors, PD98059 (a selective inhibitor of ERK), SP600125 (a selective inhibitor of JNK), and SB203580 (a selective inhibitor of p38). TNF-α and IL-6 assays were performed using enzyme-linked immunosorbent assay kits. The phosphorylation levels of p38 and nuclear factor kappa B p65 (NF-κB p65) were examined by western blot. RESULTS: Naringin or SB203580 pretreatment significantly suppressed the secretion of TNF-α and IL-6 induced by titanium particles in fibroblasts, while inhibition of ERK or JNK pathways showed no effect on production of TNF-α and IL-6. Moreover, naringin inhibited Ti particle-induced phosphorylation of p38 and p65. CONCLUSIONS: These results indicated that naringin could inhibit Ti particle-induced inflammation in fibroblasts by inhibiting p38 MAPK/NF-κB p65 activity and might be a potential drug for the treatment of inflammatory periprosthetic osteolysis after arthroplasty.

8.
BMC Genomics ; 21(1): 431, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32586283

RESUMO

BACKGROUND: The low genetic diversity of Upland cotton limits the potential for genetic improvement. Making full use of the genetic resources of Sea-island cotton will facilitate genetic improvement of widely cultivated Upland cotton varieties. The chromosome segments substitution lines (CSSLs) provide an ideal strategy for mapping quantitative trait loci (QTL) in interspecific hybridization. RESULTS: In this study, a CSSL population was developed by PCR-based markers assisted selection (MAS), derived from the crossing and backcrossing of Gossypium hirsutum (Gh) and G. barbadense (Gb), firstly. Then, by whole genome re-sequencing, 11,653,661 high-quality single nucleotide polymorphisms (SNPs) were identified which ultimately constructed 1211 recombination chromosome introgression segments from Gb. The sequencing-based physical map provided more accurate introgressions than the PCR-based markers. By exploiting CSSLs with mutant morphological traits, the genes responding for leaf shape and fuzz-less mutation in the Gb were identified. Based on a high-resolution recombination bin map to uncover genetic loci determining the phenotypic variance between Gh and Gb, 64 QTLs were identified for 14 agronomic traits with an interval length of 158 kb to 27 Mb. Surprisingly, multiple alleles of Gb showed extremely high value in enhancing cottonseed oil content (SOC). CONCLUSIONS: This study provides guidance for studying interspecific inheritance, especially breeding researchers, for future studies using the traditional PCR-based molecular markers and high-throughput re-sequencing technology in the study of CSSLs. Available resources include candidate position for controlling cotton quality and quantitative traits, and excellent breeding materials. Collectively, our results provide insights into the genetic effects of Gb alleles on the Gh, and provide guidance for the utilization of Gb alleles in interspecific breeding.

9.
Plant Biotechnol J ; 2020 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-32558152

RESUMO

Drought resistance (DR) is a complex trait that is regulated by a variety of genes. Without comprehensive profiling of DR-related traits, the knowledge of the genetic architecture for DR in cotton remains limited. Thus, there is a need to bridge the gap between genomics and phenomics. In this study, an automatic phenotyping platform (APP) was systematically applied to examine 119 image-based digital traits (i-traits) during drought stress at the seedling stage, across a natural population of 200 representative upland cotton accessions. Some novel i-traits, as well as some traditional i-traits, were used to evaluate the DR in cotton. The phenomics data allowed us to identify 390 genetic loci by genome-wide association study (GWAS) using 56 morphological and 63 texture i-traits. DR-related genes, including GhRD2, GhNAC4, GhHAT22 and GhDREB2, were identified as candidate genes by some digital traits. Further analysis of candidate genes showed that Gh_A04G0377 and Gh_A04G0378 functioned as negative regulators for cotton drought response. Based on the combined digital phenotyping, GWAS analysis and transcriptome data, we conclude that the phenomics dataset provides an excellent resource to characterize key genetic loci with an unprecedented resolution which can inform future genome-based breeding for improved DR in cotton.

10.
Plant Biotechnol J ; 2020 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-32438486

RESUMO

The CRISPR/Cas9 and Cas12a (Cpf1) tools have been used on a large scale for genome editing. A new effector with a single nuclease domain, a relatively small size, low-frequency off-target effects and cleavage capability under high temperature has been recently established and designated CRISPR/Cas12b (C2c1). Cas12b has also shown temperature inducibility in mammalian systems. Therefore, this system is potentially valuable for editing the genomes of plant species, such as cotton, that are resistant to high temperatures. Using this new system, mutants of upland cotton were successfully generated following Agrobacterium-mediated genetic transformation under a range of temperatures. Transformants (explants infected by Agrobacterium) exposed to 45 °C for 4 days showed the highest editing efficiency. No off-target mutation was detected by whole-genome sequencing. Genome edits by AacCas12b in T0 generation were faithfully passed to the T1 generation. Taken together, CRISPR/Cas12b is therefore an efficient and precise tool for genome editing in cotton plants.

11.
J Exp Bot ; 71(18): 5615-5630, 2020 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-32443155

RESUMO

Cold stress is a key environmental factor that affects plant development and productivity. In this study, RNA-seq in cotton following cold-stress treatment resulted in the identification of 5239 differentially expressed genes (DEGs) between two cultivars with differing sensitivity to low temperatures, among which GhKCS13 was found to be involved in the response. Transgenic plants overexpressing GhKCS13 showed increased sensitivity to cold stress. KEGG analysis of 418 DEGs in both GhKCS13-overexpressing and RNAi lines after treatment at 4 °C indicated that lipid biosynthesis and linoleic acid metabolism were related to cold stress. ESI-MS/MS analysis showed that overexpression of GhKCS13 led to modifications in the composition of sphingolipids and glycerolipids in the leaves, which might alter the fluidity of the cell membrane under cold conditions. In particular, differences in levels of jasmonic acid (JA) in GhKCS13 transgenic lines suggested that, together with lysophospholipids, it might mediate the cold-stress response. Our results suggest that overexpression of GhKCS13 probably causes remodeling of lipids in the endoplasmic reticulum and biosynthesis of lipid-derived JA in chloroplasts, which might account for the increased sensitivity to cold stress in the transgenic plants. Complex interactions between lipid components, lipid signaling molecules, and JA appear to determine the response to cold stress in cotton.

12.
Neurosci Lett ; 729: 134946, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32278027

RESUMO

Neuropathic pain is defined as a chronic pain. It could be resulted from a lesion of nervous systems. MicroRNAs have been reported to modulate multiple genes and pathways in neuropathic pain and neuroinflammation. Therefore, identifying the potential expression patterns of microRNAs under neuropathic pain conditions is significant. miR-128-3p has been identified in many cancers. But, its biological role in neuropathic pain progression remains poorly known. Currently, we aimed to explore the possible functions of miR-128-3p in the modulation of neuropathic pain. We displayed in the CCI rats, miR-128-3p was greatly down-regulated in the spinal cord tissues and the isolated microglias. Subsequently, LV-miR-128-3p could attenuate CCI-triggered mechanical allodynia and thermal hyperalgesia. Meanwhile, some common pro-inflammatory cytokines were significantly reduced while anti-inflammatory cytokine IL-10 was increased by miR-128-3p. Here, in the current investigation, by utilizing dual-luciferase reporter assays, ZEB1 was proved as a direct target of miR-128-3p. LV-miR-128-3p significantly repressed ZEB1 expression in microglia of CCI rats. Moreover, ZEB1 was reported to be increased in CCI rats. miR-128-3p rescued the effects of ZEB1 on neuropathic pain progression via inhibiting neuroinflammation. Taken these together, we implied miR-128-3p alleviated the progression of neuropathic pain via modulating ZEB1.

13.
Plant Mol Biol ; 103(1-2): 211-223, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32172495

RESUMO

The mitogen-activated protein kinase (MAPK) cascade pathway, which has three components, MAP3Ks, MKKs and MPKs, is involved in diverse biological processes in plants. In the current study, MAPK cascade genes were identified in three cotton species, based on gene homology with Arabidopsis. Selection pressure analysis of MAPK cascade genes revealed that purifying selection occurred among the cotton species. Expression pattern analysis showed that some MAPK cascade genes differentially expressed under abiotic stresses and phytohormones treatments, and especially under drought stress. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments showed extensive interactions between different MAPK cascade proteins. Virus-induced gene silencing (VIGS) assays showed that some MAPK cascade modules play important roles in the drought stress response, and the GhMAP3K14-GhMKK11-GhMPK31 signal pathway was demonstrated to regulate drought stress tolerance in cotton. This study provides new information on the function of MAPK cascade genes in the drought response, and will help direct molecular breeding for improved drought stress tolerance in cotton.


Assuntos
Gossypium/genética , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Aclimatação/genética , Secas , Perfilação da Expressão Gênica , Genes de Plantas , Genoma de Planta , Gossypium/enzimologia , Gossypium/fisiologia , Filogenia
14.
Plant Physiol ; 183(1): 236-249, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32139477

RESUMO

Calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK)-mediated calcium signaling has been widely reported to function in plant development and various stress responses, particularly in ion homeostasis. Sugars are the most important primary metabolites, and thus sugar homeostasis requires precise regulation. Here, we describe a CBL2-CIPK6-Tonoplast-Localized Sugar Transporter2 (TST2) molecular module in cotton (Gossypium hirsutum) that regulates plant sugar homeostasis, in particular Glc homeostasis. GhCIPK6 is recruited to the tonoplast by GhCBL2 and interacts with the tonoplast-localized sugar transporter GhTST2. Overexpression of either GhCBL2, GhCIPK6, or GhTST2 was sufficient to promote sugar accumulation in transgenic cotton, whereas RNAi-mediated knockdown of GhCIPK6 expression or CRISPR-Cas9-mediated knockout of GhTST2 resulted in significantly decreased Glc content. Moreover, mutation of GhCBL2 or GhTST2 in GhCIPK6-overexpressing cotton reinstated sugar contents comparable to wild-type plants. Heterologous expression of GhCIPK6 in Arabidopsis (Arabidopsis thaliana) also promoted Glc accumulation, whereas mutation of AtTST1/2 in GhCIPK6-overexpressing Arabidopsis similarly reinstated wild-type sugar contents, thus indicating conservation of CBL2-CIPK6-TST2-mediated sugar homeostasis among different plant species. Our characterization of the molecular players behind plant sugar homeostasis may be exploited to improve sugar contents and abiotic stress resistance in plants.

15.
Artigo em Inglês | MEDLINE | ID: mdl-32017125

RESUMO

The cotton fibre serves as a valuable experimental system to study cell wall synthesis in plants, but our understanding of the genetic regulation of this process during fibre development remains limited. We performed a genome-wide association study (GWAS) and identified 28 genetic loci associated with fibre quality in allotetraploid cotton. To investigate the regulatory roles of these loci, we sequenced fibre transcriptomes of 251 cotton accessions and identified 15 330 expression quantitative trait loci (eQTL). Analysis of local eQTL and GWAS data prioritised 13 likely causal genes for differential fibre quality in a transcriptome-wide association study (TWAS). Characterisation of distal eQTL revealed unequal genetic regulation patterns between two subgenomes, highlighted by an eQTL hotspot (Hot216) that established a genome-wide genetic network regulating the expression of 962 genes. The primary regulatory role of Hot216, and specifically the gene encoding a KIP-related protein, was found to be the transcriptional regulation of genes responsible for cell wall synthesis, which contributes to fibre length by modulating the developmental transition from rapid cell elongation to secondary cell wall synthesis. This study uncovered the genetic regulation of fibre-cell development and revealed the molecular basis of the temporal modulation of secondary cell wall synthesis during plant cell elongation.

16.
Artigo em Inglês | MEDLINE | ID: mdl-32017357

RESUMO

This study was focused on dual effects of 20 wt % hydrochloric acid etching treatment on cell responses and mechanical properties of a porous titanium with controllable open-porous structure. The results show that the acid etching induced the formation of a rough surface on the porous titanium, resulting in a remarkable improvement of the MG-63 osteoblasts adhesion and proliferation on the porous titanium. The surface roughness is found to be mainly dependent on the etching time. As increasing etching time, the surface roughness exhibited a noticeable rise. After etching for 90 min, the best cell response was achieved on the rough surface with a roughness value of 3.7 mm. However, the acid etching treatment displayed a negative effect on the porous titanium strength, and the yield strength was reduced down to 106 MPa as the etching time increased to 150 min. Taking both the cell responses and strength into account, an optimal etching time was determined as 60 min by experiments.

17.
BMC Musculoskelet Disord ; 21(1): 119, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32093650

RESUMO

BACKGROUD: The osteotomy of the posterolateral overhanging part (PLOP) of the greater trochanter via posterior approach has been used for the hip arthroplasty for decades with good results. However, the osteotomy method remains undefined and the precise adjacent structures around PLOP have not been reported. The purpose of this study was to present a modified PLOP osteotomy approach and perform a detailed study of the topographic and surgical anatomy of the PLOP. METHODS: The peri-PLOP soft tissue and the bony parameters were measured using 10 cadavers with 20 hips and 20 skeletal hip specimens, respectively. RESULTS: A 1.8-cm vertical osteotomy did not jeopardize the femoral neck, and a 1.8-cm wide bone block did not damage the insertions of the short external rotators. The average distances between the most distal branch of the superior gluteal nerve/artery and the 1.8-cm point of the greater trochanter were 5.70 ± 0.66 cm and 6.33 ± 0.56 cm, respectively. CONCLUSION: For osteotomy of the PLOP, we suggested that the width of the upper side from the lateral to medial greater trochanter should be 1.8 cm, depth of vertical osteotomy should be 1.8 cm, and length of the posterior edge should be 4 cm. Obturator externus tendon should be kept within the bone block of osteotomy. The proximal extension of the gluteus medius muscle split should be limited to 5.5 cm at the 1.8 cm-point of the greater trochanter. LEVEL OF EVIDENCE: Prospective comparative study Level II.

18.
Adv Healthc Mater ; 9(3): e1901375, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31894648

RESUMO

The increasing problem of bacterial resistance to the currently effective antibiotics has resulted in the need for increasingly potent therapeutics to eradicate pathogenic microorganisms. 2D nanomaterials (2D NMs) have unique physical and chemical properties that make them attractive candidates for biomedical applications. Recently, the application of 2D NMs as antibacterial agents has attracted significant attention. Herein, a novel 2D graphene-like silicon nanosheet (GS NS) antimicrobial agent is fabricated from pristine silicon crystals by ultrasonication, which results in a highly exfoliated planar morphology and a significantly larger surface area as compared with bulk silicon. The GS NSs exhibit remarkable in vitro broad-spectrum bactericidal activity against Gram (-) Escherichia coli and Gram (+) Staphylococcus aureus because of a close interaction with the bacteria, which leads to highly efficient membrane destruction. The in vivo studies demonstrate that the local administration of GS NSs effectively mitigates implant-related infection by reducing the bacterial burden of the extracted samples and accelerating the remission of local inflammation. Based on these encouraging results, GS NSs are expected to be a useful new member of the 2D NMs family, with the potential of effectively killing pathogenic bacteria in clinical applications.

19.
Plant Cell Rep ; 39(2): 181-194, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31713664

RESUMO

KEY MESSAGE: GbWRKY1 can function as a negative regulator of ABA signaling via JAZ1 and ABI1, with effects on salt and drought tolerance. WRKY transcription factors play important roles in plant development and stress responses. GbWRKY1 was initially identified as a defense-related gene in cotton and negatively regulates the response to fungal pathogens by activating the expression of JAZ1. Here, we characterized the role of GbWRKY1, an orthologue of the Arabidopsis gene AtWRKY75, in abiotic stress (salt and drought) and established novel connection between JAZ1 and ABA signaling in Arabidopsis. GbWRKY1 is nucleus localized and its expression is significantly induced by treatment with ABA and osmotic stresses NaCl and PEG. Increased levels of expression of GbWRKY1 in transgenic Arabidopsis enhance sensitivity to salt and drought as revealed by seed germination tests and soil stress experiments. Similarly, GbWRKY1 overexpression cotton plants also display increased sensitivity to PEG treatment and drought. Expression analysis shows that the induction of two ABA responsive genes, RAB18 and RD29A by NaCl, mannitol, and ABA treatment is significantly impaired in GbWRKY1 overexpression Arabidopsis lines. GbWRKY1 overexpression Arabidopsis displays a strong ABA-insensitive phenotype at both germination and early stages of seedling development. Further genetic evidence suggested that the ABA-insensitive phenotype of GbWRKY1 overexpression Arabidopsis was dependent on JAZ1, and overexpression of JAZ1 also displayed an ABA-insensitive phenotype. In addition, yeast two hybrid and bimolecular fluorescence complementation assays showed that JAZ1 directly interacts with ABI1, a key negative regulator of ABA signaling. We, therefore, demonstrate that GbWRKY1 acts as a negative regulator of ABA signaling, through an interaction network involving JAZ1 and ABI1, to regulate salt and drought tolerance.

20.
Plant Biotechnol J ; 18(1): 45-56, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31116473

RESUMO

The base-editing technique using CRISPR/nCas9 (Cas9 nickase) or dCas9 (deactivated Cas9) fused with cytidine deaminase is a powerful tool to create point mutations. In this study, a novel G. hirsutum-Base Editor 3 (GhBE3) base-editing system has been developed to create single-base mutations in the allotetraploid genome of cotton (Gossypium hirsutum). A cytidine deaminase sequence (APOBEC) fused with nCas9 and uracil glycosylase inhibitor (UGI) was inserted into our CRISPR/Cas9 plasmid (pRGEB32-GhU6.7). Three target sites were chosen for two target genes, GhCLA and GhPEBP, to test the efficiency and accuracy of GhBE3. The editing efficiency ranged from 26.67 to 57.78% at the three target sites. Targeted deep sequencing revealed that the C→T substitution efficiency within an 'editing window', approximately six-nucleotide windows of -17 to -12 bp from the PAM sequence, was up to 18.63% of the total sequences. The 27 most likely off-target sites predicted by CRISPR-P and Cas-OFFinder tools were analysed by targeted deep sequencing, and it was found that rare C→T substitutions (average < 0.1%) were detected in the editing windows of these sites. Furthermore, whole-genome sequencing analyses on two GhCLA-edited and one wild-type plants with about 100× depth showed that no bona fide off-target mutations were detectable from 1500 predicted potential off-target sites across the genome. In addition, the edited bases were inherited to T1 progeny. These results demonstrate that GhBE3 has high specificity and accuracy for the generation of targeted point mutations in allotetraploid cotton.

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