Legionella effector LnaB is a phosphoryl-AMPylase that impairs phosphosignalling.

AMPylation is a posttranslational modification that generally modifies amino acid side chains of proteins with adenosine monophosphate (AMP)1,2. Here we report that with ATP as the ligand and actin as the host activator, the effector protein LnaB of Legionella pneumophila exhibits AMPylase activity toward the phosphoryl group of phosphoribose on PRR42-Ub that is generated by the SidE family effectors and deubiquitinases DupA/B in an E1/E2-independent ubiquitination process3-7. The product of LnaB is further hydrolyzed by an ADP-ribosyl hydrolase, MavL, to be Ub, thereby preventing accumulation of PRR42-Ub and ADPRR42-Ub and protecting the canonical ubiquitination in host cells. LnaB represents a large family of AMPylases adopting a common structural fold, which is distinct from those of the previously known AMPylases, in bacterial pathogens of more than 20 species. Moreover, LnaB also exhibits robust phosphoryl AMPylase activity toward phosphorylated residues and produces unique ADPylation modification in proteins. During infection, LnaB AMPylates the conserved phosphorylated tyrosine residues in the activation loop of the Src family kinases8,9, which dampens the host downstream phosphorylation signaling. Structural studies revealed the actin-dependent activation and catalytic mechanisms of the LnaB family of AMPylases. This study presents an unprecedented regulation and molecular mechanism in bacterial pathogenesis and protein phosphorylation.

Transcription Factor McHB7 Improves Ice Plant Drought Tolerance through ABA Signaling Pathway.

As global climate change continues, drought episodes have become increasingly frequent. Studying plant stress tolerance is urgently needed to ensure food security. The common ice plant is one of the model halophyte plants for plant stress biology research. This study aimed to investigate the functions of a newly discovered transcription factor, Homeobox 7 (HB7), from the ice plant in response to drought stress. An efficient Agrobacterium-mediated transformation method was established in the ice plant, where ectopic McHB7 expression may be sustained for four weeks. The McHB7 overexpression (OE) plants displayed drought tolerance, and the activities of redox enzymes and chlorophyll content in the OE plants were higher than the wild type. Quantitative proteomics revealed 1910 and 495 proteins significantly changed in the OE leaves compared to the wild type under the control and drought conditions, respectively. Most increased proteins were involved in the tricarboxylic acid cycle, photosynthesis, glycolysis, pyruvate metabolism, and oxidative phosphorylation pathways. Some were found to participate in abscisic acid signaling or response. Furthermore, the abscisic acid levels increased in the OE compared with the wild type. McHB7 was revealed to bind to the promoter motifs of Early Responsive to Dehydration genes and abscisic acid-responsive genes, and protein-protein interaction analysis revealed candidate proteins responsive to stresses and hormones (e.g., abscisic acid). To conclude, McHB7 may contribute to enhance plant drought tolerance through abscisic acid signaling.

Discovery of Trametinib as an orchestrator for cytoskeletal vimentin remodeling.

The dynamic remodeling of the cytoskeletal network of vimentin intermediate filaments network supports various cellular functions, including cell morphology, elasticity, migration, organelle localization, and resistance against mechanical or pathological stress. Currently available chemicals targeting vimentin predominantly induce network reorganization and shrinkage around the nucleus. Effective tools for long-term manipulation of vimentin network dispersion in living cells are still lacking, limiting in-depth studies on vimentin function and potential therapeutic applications. Here, we verified that a commercially available small molecule, Trametinib, is capable of inducing spatial spreading of the cellular vimentin network without affecting its transcriptional or translational regulation. Further evidence confirmed its low cytotoxicity and similar effects on different cell types. Importantly, Trametinib has no impact on the other two cytoskeletal systems, actin filaments and the microtubule network. Moreover, Trametinib regulates vimentin network dispersion rapidly and efficiently, with effects persisting for up to 48 h after drug withdrawal. We also ruled out the possibility that Trametinib directly affects the phosphorylation level of vimentin. In summary, we identified an unprecedented regulator, Trametinib, capable of spreading the vimentin network toward the cell periphery, and thus complemented the existing repertoire of vimentin remodeling drugs in the field of cytoskeletal research.

Experimental Study on the Erosion-Corrosion Characteristics of Desulfurization Slurry on Stainless Steel Pipe Materials.

The recovery of low-grade waste heat from power plants greatly benefits energy conservation and emission reduction during electricity generation, while the waste heat utilization directly from desulfurization slurry is a significantly promising method to deeply recover such low-grade energy and has been developed in practical application. However, the pipe materials are subjected to erosion and corrosion challenges due to the high level of solid compositions and the presence of harmful ions, such as Cl-1, which requires further evaluation under the condition of slurry heat exchange. The present study aimed at an experimental study on the erosion-corrosion characteristics of desulfurization slurry on three types of stainless steel, including type 304, 316L, and 2205. Both mass loss and micromorphology features were analyzed with possible mechanisms elucidated. The erosion-corrosion rate is weak at low temperatures, while the increase in the slurry temperature clearly promotes its rate. The influence of the temperature on the corrosion resistance of 304 is much greater than that of 2205. With an increase in duration time, the weight loss rate of stainless steel in the desulfurization slurry declines, and the changing trend of metal mass slightly slows down. The present study offers a better understanding of the erosion-corrosion behaviors of three types of stainless steel under flow and heat transfer conditions of a desulfurization slurry.

THEMIS is a substrate and allosteric activator of SHP1, playing dual roles during T cell development.

THEMIS plays an indispensable role in T cells, but its mechanism of action has remained highly controversial. Using the systematic proximity labeling methodology PEPSI, we identify THEMIS as an uncharacterized substrate for the phosphatase SHP1. Saturated mutagenesis assays and mass spectrometry analysis reveal that phosphorylation of THEMIS at the evolutionally conserved Tyr34 residue is oppositely regulated by SHP1 and the kinase LCK. Similar to THEMIS-/- mice, THEMISY34F/Y34F knock-in mice show a significant decrease in CD4 thymocytes and mature CD4 T cells, but display normal thymic development and peripheral homeostasis of CD8 T cells. Mechanistically, the Tyr34 motif in THEMIS, when phosphorylated upon T cell antigen receptor activation, appears to act as an allosteric regulator, binding and stabilizing SHP1 in its active conformation, thus ensuring appropriate negative regulation of T cell antigen receptor signaling. However, cytokine signaling in CD8 T cells fails to elicit THEMIS Tyr34 phosphorylation, indicating both Tyr34 phosphorylation-dependent and phosphorylation-independent roles of THEMIS in controlling T cell maturation and expansion.

Allosterically inhibited PFKL via prostaglandin E2 withholds glucose metabolism and ovarian cancer invasiveness.

Metastasis is the leading cause of high ovarian-cancer-related mortality worldwide. Three major processes constitute the whole metastatic cascade: invasion, intravasation, and extravasation. Tumor cells often reprogram their metabolism to gain advantages in proliferation and survival. However, whether and how those metabolic alterations contribute to the invasiveness of tumor cells has yet to be fully understood. Here we performed a genome-wide CRISPR-Cas9 screening to identify genes participating in tumor cell dissemination and revealed that PTGES3 acts as an invasion suppressor in ovarian cancer. Mechanistically, PTGES3 binds to phosphofructokinase, liver type (PFKL) and generates a local source of prostaglandin E2 (PGE2) to allosterically inhibit the enzymatic activity of PFKL. Repressed PFKL leads to downgraded glycolysis and the subsequent TCA cycle for glucose metabolism. However, ovarian cancer suppresses the expression of PTGES3 and disrupts the PTGES3-PGE2-PFKL inhibitory axis, leading to hyperactivation of glucose oxidation, eventually facilitating ovarian cancer cell motility and invasiveness.

Development of the nonreceptor tyrosine kinase FER-targeting PROTACs as a potential strategy for antagonizing ovarian cancer cell motility and invasiveness.

Aberrant overexpression of nonreceptor tyrosine kinase FER (Fps/Fes Related) has been reported in various ovarian carcinoma-derived tumor cells and is a poor prognosis factor for patient survival. It plays an essential role in tumor cell migration and invasion, acting concurrently in both kinase-dependent and -independent manners, which is not easily suppressed by conventional enzymatic inhibitors. Nevertheless, the PROteolysis-TArgeting Chimera (PROTAC) technology offers superior efficacy over traditional activity-based inhibitors by simultaneously targeting enzymatic and scaffold functions. Hence in this study, we report the development of two PROTAC compounds that promote robust FER degradation in a cereblon-dependent manner. Both PROTAC degraders outperform a Food and Drug Administration-approved drug, brigatinib, in ovarian cancer cell motility suppression. Importantly, these PROTAC compounds also degrade multiple oncogenic FER fusion proteins identified in human tumor samples. These results lay an experimental foundation to apply the PROTAC strategy to antagonize cell motility and invasiveness in ovarian and other types of cancers with aberrant expression of FER kinase and highlight PROTACs as a superior strategy for targeting proteins with multiple tumor-promoting functions.

Tuning charge density of chimeric antigen receptor optimizes tonic signaling and CAR-T cell fitness.

Tonic signaling of chimeric antigen receptor (CAR), i.e., the spontaneous CAR activation in the absence of tumor antigen stimulation, is considered to be a pivotal event controlling CAR-T efficacy. However, the molecular mechanism underlying the spontaneous CAR signals remains elusive. Here, we unveil that positively charged patches (PCPs) on the surface of the CAR antigen-binding domain mediate CAR clustering and result in CAR tonic signaling. For CARs with high tonic signaling (e.g., GD2.CAR and CSPG4.CAR), reducing PCPs on CARs or boosting ionic strength in the culture medium during ex vivo CAR-T cell expansion minimizes spontaneous CAR activation and alleviates CAR-T cell exhaustion. In contrast, introducing PCPs into the CAR with weak tonic signaling, such as CD19.CAR, results in improved in vivo persistence and superior antitumor function. These results demonstrate that CAR tonic signaling is induced and maintained by PCP-mediated CAR clustering. Notably, the mutations we generated to alter the PCPs maintain the antigen-binding affinity and specificity of the CAR. Therefore, our findings suggest that the rational tuning of PCPs to optimize tonic signaling and in vivo fitness of CAR-T cells is a promising design strategy for the next-generation CAR.

Addressing the Enzyme-independent tumor-promoting function of NAMPT via PROTAC-mediated degradation.

Aberrant overexpression of nicotinamide phosphoribosyltransferase (NAMPT) has been reported in a variety of tumor cells and is a poor prognosis factor for patient survival. It plays an important role in tumor cell proliferation, acting concurrently as an nicotinamide adenine dinucleotide (NAD+) synthase and, unexpectedly, as an extracellular signaling molecule for several tumor-promoting pathways. Although previous efforts to modulate NAMPT activity were limited to enzymatic inhibitors with low success in clinical studies, protein degradation offers the possibility to simultaneously disrupt NAMPT's enzyme activity and ligand capabilities. Here we report the development of two highly selective proteolysis-targeting chimeras (PROTACs) that promote NAMPT degradation in a cereblon-dependent manner. Both PROTAC degraders outperform a clinical candidate, FK866, in killing effect on hematological tumor cells. These results emphasize the importance and feasibility of applying PROTACs as a superior strategy for targeting proteins with multiple tumor-promoting functions like NAMPT, which is not easily achieved by conventional enzymatic inhibitors.

Overexpression of PagERF072 from Poplar Improves Salt Tolerance.

Extreme environments, especially drought and high salt conditions, seriously affect plant growth and development. Ethylene-responsive factor (ERF) transcription factors play an important role in salt stress response. In this study, a significantly upregulated ERF gene was identified in 84K (Populus alba × P. glandulosa), which was named PagERF072. PagERF072 was confirmed to be a nuclear-localized protein. The results of yeast two-hybrid (Y2H) assay showed that PagERF072 protein exhibited no self-activating activity, and yeast one-hybrid (Y1H) demonstrated that PagERF072 could specifically bind to GCC-box element. Under salt stress, the transgenic poplar lines overexpressing PagERF072 showed improved salt tolerance. The activities of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) in transgenic poplars were significantly increased relative to those of wild-type (WT) plants, whereas malondialdehyde (MDA) content showed an opposite trend. In addition, reactive oxygen species (ROS) was significantly reduced, and the expression levels of POD- and SOD-related genes were significantly increased in transgenic poplars under salt stress compared with WT. All results indicate that overexpression of the PagERF072 gene can improve the salt tolerance of transgenic poplars.

Genome-Wide Identification and Expression Patterns of the F-box Family in Poplar under Salt Stress.

The F-box family exists in a wide variety of plants and plays an extremely important role in plant growth, development and stress responses. However, systematic studies of F-box family have not been reported in populus trichocarpa. In the present study, 245 PtrFBX proteins in total were identified, and a phylogenetic tree was constructed on the basis of their C-terminal conserved domains, which was divided into 16 groups (A-P). F-box proteins were located in 19 chromosomes and six scaffolds, and segmental duplication was main force for the evolution of the F-box family in poplar. Collinearity analysis was conducted between poplar and other species including Arabidopsis thaliana, Glycine max, Anemone vitifolia Buch, Oryza sativa and Zea mays, which indicated that poplar has a relatively close relationship with G. max. The promoter regions of PtrFBX genes mainly contain two kinds of cis-elements, including hormone-responsive elements and stress-related elements. Transcriptome analysis indicated that there were 82 differentially expressed PtrFBX genes (DEGs), among which 64 DEGs were in the roots, 17 in the leaves and 26 in the stems. In addition, a co-expression network analysis of four representative PtrFBX genes indicated that their co-expression gene sets were mainly involved in abiotic stress responses and complex physiological processes. Using bioinformatic methods, we explored the structure, evolution and expression pattern of F-box genes in poplar, which provided clues to the molecular function of F-box family members and the screening of salt-tolerant PtrFBX genes.

FER-mediated phosphorylation and PIK3R2 recruitment on IRS4 promotes AKT activation and tumorigenesis in ovarian cancer cells.

Tyrosine phosphorylation, orchestrated by tyrosine kinases and phosphatases, modulates a multi-layered signaling network in a time- and space-dependent manner. Dysregulation of this post-translational modification is inevitably associated with pathological diseases. Our previous work has demonstrated that non-receptor tyrosine kinase FER is upregulated in ovarian cancer, knocking down which attenuates metastatic phenotypes. However, due to the limited number of known substrates in the ovarian cancer context, the molecular basis for its pro-proliferation activity remains enigmatic. Here, we employed mass spectrometry and biochemical approaches to identify insulin receptor substrate 4 (IRS4) as a novel substrate of FER. FER engaged its kinase domain to associate with the PH and PTB domains of IRS4. Using a proximity-based tagging system in ovarian carcinoma-derived OVCAR-5 cells, we determined that FER-mediated phosphorylation of Tyr779 enables IRS4 to recruit PIK3R2/p85ß, the regulatory subunit of PI3K, and activate the PI3K-AKT pathway. Rescuing IRS4-null ovarian tumor cells with phosphorylation-defective mutant, but not WT IRS4 delayed ovarian tumor cell proliferation both in vitro and in vivo. Overall, we revealed a kinase-substrate mode between FER and IRS4, and the pharmacological inhibition of FER kinase may be beneficial for ovarian cancer patients with PI3K-AKT hyperactivation.

Functional analysis of PagNAC045 transcription factor that improves salt and ABA tolerance in transgenic tobacco.

BACKGROUND: Salt stress causes inhibition of plant growth and development, and always leads to an increasing threat to plant agriculture. Transcription factors regulate the expression of various genes for stress response and adaptation. It's crucial to reveal the regulatory mechanisms of transcription factors in the response to salt stress. RESULTS: A salt-inducible NAC transcription factor gene PagNAC045 was isolated from Populus alba×P. glandulosa. The PagNAC045 had a high sequence similarity with NAC045 (Potri.007G099400.1) in P. trichocarpa, and they both contained the same conserved motifs 1 and 2, which constitute the highly conserved NAM domain at the N-terminus. Protein-protein interaction (PPI) prediction showed that PagNAC045 potentially interacts with many proteins involved in plant hormone signaling, DNA-binding and transcriptional regulation. The results of subcellular localization and transient expression in tobacco leaves confirmed the nuclear localization of PagNAC045. Yeast two-hybrid revealed that PagNAC045 protein exhibits transcriptional activation property and the activation domain located in its C-terminus. In addition, the 1063 bp promoter of PagNAC045 was able to drive GUS gene expression in the leaves and roots. In poplar leaves and roots, PagNAC045 expression increased significantly by salt and ABA treatments. Tobacco seedlings overexpressing PagNAC045 exhibited enhanced tolerance to NaCl and ABA compared to the wild-type (WT). Yeast one-hybrid assay demonstrated that a bHLH104-like transcription factor can bind to the promoter sequence of PagNAC045. CONCLUSION: The PagNAC045 functions as positive regulator in plant responses to NaCl and ABA-mediated stresses.

Induction of senescence-associated secretory phenotype underlies the therapeutic efficacy of PRC2 inhibition in cancer.

The methyltransferase Polycomb Repressive Complex 2 (PRC2), composed of EZH2, SUZ12, and EED subunits, is associated with transcriptional repression via tri-methylation of histone H3 on lysine 27 residue (H3K27me3). PRC2 is a valid drug target, as the EZH2 gain-of-function mutations identified in patient samples drive tumorigenesis. PRC2 inhibitors have been discovered and demonstrated anti-cancer efficacy in clinic. However, their pharmacological mechanisms are poorly understood. MAK683 is a potent EED inhibitor in clinical development. Focusing on MAK683-sensitive tumors with SMARCB1 or ARID1A loss, we identified a group of PRC2 target genes with high H3K27me3 signal through epigenomic and transcriptomic analysis. Multiple senescence-associated secretory phenotype (SASP) genes, such as GATA4, MMP2/10, ITGA2 and GBP1, are in this group besides previously identified CDKN2A/p16. Upon PRC2 inhibition, the de-repression of SASP genes is detected in multiple sensitive models and contributes to decreased Ki67+, extracellular matrix (ECM) reorganization, senescence associated inflammation and tumor regression even in CDKN2A/p16 knockout tumor. And the combination of PRC2 inhibitor and CDK4/6 inhibitor leads to better effect. The genes potential regulated by PRC2 in neuroblastoma samples exhibited significant enrichment of ECM and senescence associated inflammation, supporting the clinical relevance of our results. Altogether, our results unravel the pharmacological mechanism of PRC2 inhibitors and propose a combination strategy for MAK683 and other PRC2 drugs.

NAD+ supplement potentiates tumor-killing function by rescuing defective TUB-mediated NAMPT transcription in tumor-infiltrated T cells.

Although tumor-infiltrating lymphocytes (TILs) maintain their ability to proliferate, persist, and eradicate tumors, they are frequently dysfunctional in situ. By performing both whole-genome CRISPR and metabolic inhibitor screens, we identify that nicotinamide phosphoribosyltransferase (NAMPT) is required for T cell activation. NAMPT is low in TILs, and its expression is controlled by the transcriptional factor Tubby (TUB), whose activity depends on the T cell receptor-phospholipase C gamma (TCR-PLCγ) signaling axis. The intracellular level of NAD+, whose synthesis is dependent on the NAMPT-mediated salvage pathway, is also decreased in TILs. Liquid chromatography-mass spectrometry (LC-MS) and isotopic labeling studies confirm that NAD+ depletion led to suppressed glycolysis, disrupted mitochondrial function, and dampened ATP synthesis. Excitingly, both adoptive CAR-T and anti-PD1 immune checkpoint blockade mouse models demonstrate that NAD+ supplementation enhanced the tumor-killing efficacy of T cells. Collectively, this study reveals that an impaired TCR-TUB-NAMPT-NAD+ axis leads to T cell dysfunction in the tumor microenvironment, and an over-the-counter nutrient supplement of NAD+ could boost T-cell-based immunotherapy.

Genome-wide search and structural and functional analyses for late embryogenesis-abundant (LEA) gene family in poplar.

BACKGROUND: The Late Embryogenesis-Abundant (LEA) gene families, which play significant roles in regulation of tolerance to abiotic stresses, widely exist in higher plants. Poplar is a tree species that has important ecological and economic values. But systematic studies on the gene family have not been reported yet in poplar. RESULTS: On the basis of genome-wide search, we identified 88 LEA genes from Populus trichocarpa and renamed them as PtrLEA. The PtrLEA genes have fewer introns, and their promoters contain more cis-regulatory elements related to abiotic stress tolerance. Our results from comparative genomics indicated that the PtrLEA genes are conserved and homologous to related genes in other species, such as Eucalyptus robusta, Solanum lycopersicum and Arabidopsis. Using RNA-Seq data collected from poplar under two conditions (with and without salt treatment), we detected 24, 22 and 19 differentially expressed genes (DEGs) in roots, stems and leaves, respectively. Then we performed spatiotemporal expression analysis of the four up-regulated DEGs shared by the tissues, constructed gene co-expression-based networks, and investigated gene function annotations. CONCLUSION: Lines of evidence indicated that the PtrLEA genes play significant roles in poplar growth and development, as well as in responses to salt stress.

De novo synthesis and salvage pathway coordinately regulate polyamine homeostasis and determine T cell proliferation and function.

Robust and effective T cell-mediated immune responses require proper allocation of metabolic resources through metabolic pathways to sustain the energetically costly immune response. As an essential class of polycationic metabolites ubiquitously present in all living organisms, the polyamine pool is tightly regulated by biosynthesis and salvage pathway. We demonstrated that arginine is a major carbon donor and glutamine is a minor carbon donor for polyamine biosynthesis in T cells. Accordingly, the dependence of T cells can be partially relieved by replenishing the polyamine pool. In response to the blockage of biosynthesis, T cells can rapidly restore the polyamine pool through a compensatory increase in extracellular polyamine uptake, indicating a layer of metabolic plasticity. Simultaneously blocking synthesis and uptake depletes the intracellular polyamine pool, inhibits T cell proliferation, and suppresses T cell inflammation, indicating the potential therapeutic value of targeting the polyamine pool for managing inflammatory and autoimmune diseases.

Chimeric Antigen Receptor Designed to Prevent Ubiquitination and Downregulation Showed Durable Antitumor Efficacy.

Clinical evidence suggests that poor persistence of chimeric antigen receptor-T cells (CAR-T) in patients limits therapeutic efficacy. Here, we designed a CAR with recyclable capability to promote in vivo persistence and to sustain antitumor activity. We showed that the engagement of tumor antigens induced rapid ubiquitination of CARs, causing CAR downmodulation followed by lysosomal degradation. Blocking CAR ubiquitination by mutating all lysines in the CAR cytoplasmic domain (CARKR) markedly repressed CAR downmodulation by inhibiting lysosomal degradation while enhancing recycling of internalized CARs back to the cell surface. Upon encountering tumor antigens, CARKR-T cells ameliorated the loss of surface CARs, which promoted their long-term killing capacity. Moreover, CARKR-T cells containing 4-1BB signaling domains displayed elevated endosomal 4-1BB signaling that enhanced oxidative phosphorylation and promoted memory T cell differentiation, leading to superior persistence in vivo. Collectively, our study provides a straightforward strategy to optimize CAR-T antitumor efficacy by redirecting CAR trafficking.

Effects of Ultrasonic Bending Vibration Introduced by an L-Shaped Ultrasonic Rod on the Microstructure and Properties of a 1060 Aluminum Alloy Strip Formed by Twin-Roll Casting.

In order to achieve the industrial application of ultrasonic energy in the continuous casting and rolling production of aluminum alloy, a new type of L-shaped ultrasonic rod was used to introduce an ultrasonic bending vibration into the aluminum melt in the launder during the horizontal twin-roll continuous casting and rolling process of a 1060 aluminum alloy. The effects of the ultrasonic bending vibration on the microstructure and properties of the 1060 aluminum alloy cast rolling strip and its subsequent cold rolling strip were studied experimentally, and the effect of the ultrasonic-assisted refining with different amounts of Al-Ti-B refiner was explored. The results show that under the same addition amount of Al-Ti-B refiner, the ultrasonic bending vibration can refine the grains of the cast rolling strip, make the distribution of precipitates more uniform, reduce the slag inclusion defects, and improve the mechanical properties to a certain extent. The microstructure and properties of the ultrasonic cast rolling strip with 0.18 wt% Al-Ti-B refiner or 0.12 wt% Al-Ti-B refiner are better than those of the conventional cast rolling strip, but the microstructure and properties of the ultrasonic cast rolling strip with 0.09 wt% Al-Ti-B refiner are slightly worse than those of the conventional cast rolling strip. Moreover, after cold rolling, the effect of the ultrasonic bending vibration on the improvement of the microstructure and properties of the aluminum alloy strip is inherited. A comprehensive analysis shows that the use of ultrasonic energy in this paper cannot completely replace the effect of the Al-Ti-B refiner, but it can reduce the addition amount of the Al-Ti-B refiner by 1/3.

CRISPR system: Discovery, development and off-target detection.

As a revolutionary gene editing tool based on the adaptive immune defense mechanism of bacteria and archaea against exogenous DNA invasion, CRISPR/Cas system shows many remarkable characteristics over ZFNs and TALENs. However, off-target effect remains as one of the major imperfection of CRISPR/Cas system, hindering its further application in translational research. In this review, we highlight major breakthrough cross the development/application of this powerful toolkit, and summarize feasible methods for detecting potential off-target effects during genetic manipulation. We hope this review will assist scientists for accurate genomic editing in their future research.