Search

Myostatin is a member of the TGF-β superfamily and functions as a negative regulator for skeletal muscle development and growth. It has become the most targeted gene in aquaculture that used for selective breeding. Previous studies involved in genome editing in several fish species confirmed that CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) system was highly efficient with lower off-target effect, however, no reports were raised in Culter alburnus. In this study, we employed CRISPR/Cas9 gene editing system to successfully disrupt mstn gene by co-injection with Cas9 protein and the targeted sgRNA in C. alburnus. Various Indel mutations were obtained with 82% knockout efficiency in the F0 generation by PCR sequencing. In addition, mutations in mstn that induced by CRISPR/Cas9 were detected in the F1 generation by individually mating the wild-type female with the F0 generation of mstn-KO male at sexual maturity. More importantly, the body weight and length were significantly elevated in mstn ± group when compared to those of the control. As expected in mstn ± group, the expression level of mstn was sharply reduced, whereas a slight increase was observed in two growth-related genes (myod and myog). Moreover, higher numbers of muscle fibers were observed in mstn ± group, meaning that growth performance in mstn ± individuals might be represented by increasing the number of muscle fibers. Taken together, our current study successfully obtained a site-specific modification of mstn using CRISPR/Cas9 technology, and these results provided a new insight for facilitating topmouth culter genetic studies and breeding.

The topmouth culter (Culter alburnus) is at the top of the food chain and plays an important role in the maintenance of freshwater ecosystem stability. It is also a commercially important aquaculture fish species in China. To establish conservation strategies and breeding programs, a total of 16 polymorphic microsatellite loci were isolated from transcriptome-derived sequences of C. alburnus. Characterization of these loci was assessed against 1 wild and 3 cultured populations in Huzhou, China. The number of alleles per locus ranged from 3 to 10 with an average of 5.25. A bottleneck effect was observed in all 4 populations; The ChangXing population showed moderate genetic differentiation when compared with other populations (FST values, 0.0695–0.0788). The microsatellite loci would be useful for further studies on population genetic diversity, genetic structure, and assessments of the artificial propagation release effect of C. alburnus.

Abstract Background The introduction of non-native species is a primary driver of biodiversity loss in freshwater ecosystems. The redclaw crayfish (Cherax quadricarinatus) is a freshwater species that exhibits tolerance to hypoxic stresses, fluctuating temperatures, high ammonia concentration. These hardy physiological characteristics make C. quadricarinatus a popular aquaculture species and a potential invasive species that can negatively impact tropical and subtropical ecosystems. Investigating the genomic basis of environmental tolerances and immune adaptation in C. quadricarinatus will facilitate the development of management strategies of this potential invasive species. Results We constructed a chromosome-level genome of C. quadricarinatus by integrating Nanopore and PacBio techniques. Comparative genomic analysis suggested that transposable elements and tandem repeats drove genome size evolution in decapod crustaceans. The expansion of nine immune-related gene families contributed to the disease resistance of C. quadricarinatus. Three hypoxia-related genes (KDM3A, KDM5A, HMOX2) were identified as being subjected to positive selection in C. quadricarinatus. Additionally, in vivo analysis revealed that upregulating KDM5A was crucial for hypoxic response in C. quadricarinatus. Knockdown of KDM5A impaired hypoxia tolerance in this species. Conclusions Our results provide the genomic basis for hypoxic tolerance and immune adaptation in C. quadricarinatus, facilitating the management of this potential invasive species. Additionally, in vivo analysis in C. quadricarinatus suggests that the role of KDM5A in the hypoxic response of animals is complex.

Abstract Acrossocheilus fasciatus (Cypriniformes, Cyprinidae) is emerged as a newly commercial stream fish in the south of China with high economic and ornamental value. In this study, a chromosome-level reference genome of A. fasciatus was assembled using PacBio, Illumina and Hi-C sequencing technologies. As a result, a high-quality genome was generated with a size of 879.52 Mb (accession number: JAVLVS000000000), scaffold N50 of 32.7 Mb, and contig N50 of 32.7 Mb. The largest and smallest scafford was 60.57 Mb and 16 kb, respectively. BUSCO analysis showed a completeness score of 98.3%. Meanwhile, the assembled sequences were anchored to 25 pseudo-chromosomes with an integration efficiency of 96.95%. Additionally, we found approximately 390.91 Mb of repetitive sequences that accounting for 44.45% of the assembled genome, and predicted 24,900 protein-coding genes. The available genome reported in the present study provided a crucial resource to further investigate the regulation mechanism of genetic diversity, sexual dimorphism and evolutionary histories.

【Objective】Xenocypris davidi is a freshwater economic fish, which feeds on humus, organic debris, plankton, and benthic animals, is one of the main fish for stock enhancement in Zhejiang Province. The research is aims to understand the impact of artificial breeding on the genetic diversity of X. davidi, and to provide basic data for the design and implementation of stock enhancement strategies for X. davidi in natural waters.【Method】D-loop region of mitochondrial DNA (mtDNA) of four cultured X. davidi populations from Changxing, Balidian, Shuangpu in Zhejiang Provice and Liling in Hunan Province were amplified and sequenced. The genetic diversity of the four populations was studied by sequence analysis.【Result】The mitochondrial D-loop sequence length of the X. davidi was 1 038-1 093 bp, and the base A+T content (65.3%) was significantly higher than the C+G content (34.7%), the average transitions and transversions ratio (TS/TV) was 4.6. A total of 101 mutation sites including 97 reduced information sites were detected in 128 D-loop sequences of X. davidi, and defining 19 haplotypes. Among them, the haplotype numbers of Changxing, Shuangpu, Balidian, and Liling populations were 5, 12, 4, and 2, respectively. The haplotype diversity (h) ranged from 0.226 to 0.915, and the nucleotide diversity (π) ranged from 0.00640 to 0.01433. The genetic distance between different breeding populations was 0.03782-0.88756, the genetic differentiation coefficient among populations was 0.78903 (P < 0.01), among which the degree of genetic difference between Changxing and Balidian populations was the lowest, and that of Shuangpu and Liling populations was the highest, and the genetic variation mainly occurred among populations.【Conclusion】There are certain differences in genetic diversity among the four cultured X. davidi populations. The results of the genetic variation and population structure of X. davidi will provide reference data for the comparative study on the genetic diversity of the X. davidi, and contribute to the resource protection.

Objective: To report a case of da Vinci robotic-assisted radical resection of hilar cholangiocarcinoma. This case is shared to discuss the techniques, methods and ideas for the treatment of hilar cholangiocarcinoma. Methods: The patient was diagnosed with hilar cholangiocarcinoma type II through preoperative examination, and surgical planning was performed after 3D reconstruction. After adequate preoperative preparation, a robotic-assisted Taj Mahal procedure was performed to radically resect the hilar cholangiocarcinoma, and then perihilar resection, hilar cholangioplasty and digestive tract reconstruction were completed (Informed consent was obtained from the patient). Results: The surgical procedure went smoothly and the postoperative recovery was good, with no tumor recurrence at 6 months of follow-up. Conclusion: The da Vinci robotic-assisted Taj Mahal for hilar cholangiocarcinoma was performed safely and effectively on patients with common hepatic duct nodule and dilatation of the intrahepatic bile ducts. Meanwhile, robotic-assisted systems have a broad application prospect and are bound to give a great impetus to surgical treatment in the future.

Topmouth culter (Culter alburnus) is an economically important freshwater fish with high nutritional value. However, its potential genetic advantages have not been fully exploited. Therefore, we aimed to determine the genome sequence of C. alburnus and examine quantitative trait loci (QTLs) related to major economic traits. The results showed that 24 pseudochromosomes were anchored by 914.74 Mb of the C. alburnus genome sequence. De novo sequencing identified 31,279 protein-coding genes with an average length of 8507 bp and average coding sequ ence of 1115 bp. In addition, a high-density genetic linkage map consisting of 24 linkage groups was constructed based on 353,532 high-quality single nucleotide polymorphisms and 4,710 bin markers. A total of 28 QTLs corresponding to 11 genes, 26 QTLs corresponding to 11 genes, and 12 QTLs corresponding to 5 genes were identified for sex, intermuscular spine number and body weight traits, respectively. In this study, we assembled an accurate and nearly complete genome of C. alburnus by combining Illumina, PacBio, and high-throughput Chromosome conformation capture (Hi-C) technologies. In addition, we identified QTLs that explained variances in intermuscular spine number, body weight, and sex differences in C. alburnus. These genetic markers or candidate genes associated with growth traits provide a basis for marker-assisted selection in C. alburnus.

The internal gear pump is simple in structure, small in size and light in weight. It is an important basic component that supports the development of hydraulic system with low noise. However, its working environment is harsh and complex, and there are hidden risks related to reliability and exposure of acoustic characteristics over the long term. In order to meet the requirements of reliability and low noise, it is very necessary to make models with strong theoretical value and practical significant to accurately monitor health and predict the remaininglife of the internal gear pump. This paper proposed a multi-channel internal gear pump health status management model based on Robust-ResNet. Robust-ResNet is an optimized ResNet model based on a step factor h in the Eulerian approach to enhance the robustness of the ResNet model. This model was a two-stage deep learning model that classified the current health status of internal gear pumps, and also predicted the remaining useful life (RUL) of internal gear pumps. The model was tested in an internal gear pump dataset collected by the authors. The model was also proven to be useful in the rolling bearing data from Case Western Reserve University (CWRU). The accuracy results of health status classification model were 99.96% and 99.94% in the two datasets. The accuracy of RUL prediction stage in the self-collected dataset was 99.53%. The results demonstrated that the proposed model achieved the best performance compared to other deep learning models and previous studies. The proposed method was also proven to have high inference speed; it could also achieve real-time monitoring of gear health management. This paper provides an extremely effective deep learning model for internal gear pump health management with great application value.

Friction is an inherent nonlinear disturbance that can lead to creeping, jitter, and decreased tracking precision in an electro-hydraulic servo system. In this paper, the LuGre friction model is used to describe the dynamic and static characteristics of the friction force of a servo system comprehensively. Accurate identification of model parameters is key to implementing friction compensation. However, traditional genetic identification algorithms have the shortcomings of a premature solution, slow convergence, and poor accuracy. To address these shortcomings, this paper proposes an improved adaptive genetic identification algorithm. The proposed algorithm selects evolutionary processes adaptively according to the population concentration in the initial stage of population evolution. Moreover, it adjusts the crossover probability and the mutation probability to identify a local optimum accurately and converge to the global optimum rapidly. During the late stage of population evolution, the accuracy of the global optimal solution can be improved by reducing the search range of identification parameters. The simulation results show that the relative error of the model parameter values identified by the proposed algorithm is reduced to less than 1% and the convergence speed is faster. Compared with the existing traditional genetic algorithm and adaptive genetic algorithm, the overall performance of the proposed method is better. This study provides a feasible and highly accurate identification method for parameter identification of friction models used in electro-hydraulic servo systems.

Redclaw crayfish (Cherax quadricarinatus) was introduced to China many years ago. In recent years, a breeding boom for C. quadricarinatus has been set off in China due to a breakthrough in key technology of seedling breeding. The size and growth rate of C. quadricarinatus vary greatly between female and male individuals, usually the size and growth rate of male individuals are bigger than that of female individuals. There is usually a certain linkage relationship between the sex traits of crustaceans and their own nutrition. In order to explore the linkage relationship between the sex traits of C. quadricarinatus and its nutritional components, this study measured and analyzed the muscle nutritional components of female and male individuals. The results showed that the meat yield rate of male individuals was significantly higher than that of females (p < 0.05), and the crude fat content was significantly lower than that for females (p < 0.05). The ratios of essential amino acids to total amino acids for females and males were 39.61% and 38.49%, respectively. The ratios of essential amino acids to non-essential amino acids were 79.69% and 75.66%, respectively, which far exceed FAO/WHO standards and both belong to high-quality protein. The total amount of flavor amino acids of male individuals was significantly higher than that of female individuals (p < 0.05). The total amount of polyunsaturated fatty acids and the polyunsaturated fatty acid eicosapentaenoic acid of males are both significantly higher than that of females (p < 0.05). Studies have shown that there are certain differences in nutrition between male and female individuals. Compared with female individuals, the meat yield rate, crude protein content, and edible value of the muscles of male individuals is higher.

The raw signals produced by internal gear pumps are susceptible to noises brought on by mechanical vibrations and the surrounding environment, and the sample count collected during the various operating periods is not distributed evenly. Accurately diagnosing faults in internal gear pumps is significantly complicated by these factors. In light of these issues, accelerated life testing was performed in order to collect signals from an internal gear pump during various operating periods. Based on the architecture of a convolutional auto-encoder network, preprocessing of the signals in the various operating periods was performed to suppress noise and enhance operating period-representing features. Thereafter, variational mode decomposition was utilized to decompose the preprocessed signal into multiple intrinsic mode functions, and the multi-scale permutation entropy value was extracted for each intrinsic mode function to form a feature set. The feature set was subsequently divided into a training set and a test set, with the training set being trained to utilize a particle swarm optimization–least squares support vector machine network. For pattern recognition, the test set samples were fed into the trained model. The results demonstrated a 99.2% diagnostic accuracy. Compared to other methods of fault diagnosis, the proposed method is more effective and accurate.

Fiber-Wireless (FiWi) access network is a promising network architecture to provide the constantly available connections for the collaboration of objects in the Internet of Things (IoT). The network virtualization is dominating the evolution of FiWi, i.e., virtualized FiWi access network, which shields the difference between wireless and optical subnetworks and enables the customized transmission of different types of the IoT services in a common substrate network. However, the differences between optical and wireless subnetworks pose severe obstacle on their interoperability of resource allocation. In this paper, we focus on the survivable virtual network embedding (SVNE) in the FiWi access network for the purpose of the robust IoT service provisioning even in the scenario of network component failure. Each type of the IoT service is carried on one virtual network (VN), which is characterized by not only computing the resource and bandwidth resource demands but also the connection availability requirement. A connection availability model is proposed as an indicator of the network survivability and the IoT service robustness. We aim to allocate the resource for each VN with the resource cost minimized. The optimization problem of resource allocation is formulated in the integer linear programming (ILP) model that is solved for the optimal solution in a small-scale network. A heuristic algorithm is further put forward to facilitate the use of the proposed SVNE mechanism in a large-scale network. The simulation results verify that the proposed SVNE mechanism gains significant advantages in lower resource redundancy and higher acceptance ratio of VNs.

Research in psychology, pedagogy, and physiology has shown that learning a second language will be a great benefit to preschool children. Compared with adults, children have more advantages in learning, such as better pronunciation and intonation. However, because of children’s inattention, the effects of the second language education in many preschools are not ideal in China. To make children learn the second language effectively, it should be integrated into their daily lives and in line with their interests. In this paper, an educational robot system with object recognition technology is introduced, which aims to provide innovative second language learning services for preschool children in China. The proposed system combines object recognition and projection with English teaching and makes objects in daily life more interesting with projected animation to attract children’s attention. On the projection screen, children can interact with a robot by touch or movement and can easily trigger more interactive effects. To evaluate the effectiveness of the proposed system, we conducted an experiment, and the results showed that the system can improve the language learning efficiency for the Chinese preschool children by interacting with objects under proper guidance.

Mechanical equipment failure may cause massive economic and even life loss. Therefore, the diagnosis of the failures of machine parts in time is crucial. The rolling bearings are one of the most valuable parts, which have attracted the focus of fault diagnosis. Many successful rolling bearing fault diagnoses have been made based on machine learning and deep learning. However, most diagnosis methods still rely on complex signal processing and artificial features, bringing many costs to the deployment and migration of diagnostic models. This paper proposes an end-to-end continuous/discontinuous feature fusion method for rolling bearing fault diagnosis (C/D-FUSA). This method comprises long short-term memory (LSTM), convolutional neural networks (CNN) and attention mechanism, which automatically extracts the continuous and discontinuous features from vibration signals for fault diagnosis. We also propose a contextual-dependent attention module for the LSTM layers. We compare the method with the other simpler deep learning methods and state-of-the-art methods in rolling bearing fault data sets with different sample rates. The results show that our method is more accurate than the other methods with real-time inference. It is also easy to be deployed and trained in a new environment.

Sex determination/sex differentiation is determined by genetics, environmental factors, or the interactions of the two. The Transformer-2 (Tra-2) gene plays an important role in the sex determination cascade signal pathway in insects. In this study, the Tra-2 gene was isolated and characterized from the cDNA library of gonad tissues in the redclaw crayfish, Cherax quadricarinatus. Three splice variants were identified, designated as CqTra-2A, CqTra-2B, and CqTra-2C, and sequence analysis showed that they had a highly conserved RRM domain. Phylogenetic analysis was performed by the NJ method, and the results revealed that the Tra-2 protein of the redclaw crayfish was very closely related to those of Macrobrachium rosenbergii, Fenneropenaeus chinensis, and Macrobrachium nipponense. Real-time PCR analysis showed that the three isoforms were predominantly expressed in the ovary and gradually increased with embryonic development. Additionally, the expression pattern of CqTra-2 at different developmental stages was analyzed by qPCR and revealed that the phase of having a body length of 3 cm may be the key period for the sex differentiation of C. quadricarinatus. RNAi-targeting gene silencing further confirmed the function of CqTra-2 in sexual differentiation in redclaw crayfish. Our experimental data will contribute to understanding the mechanism of sex determination in crustaceans.

Abstract Background In somatic cells, homologous recombination (HR) is a rare event caused by eventual DNA double-strand breaks (DSBs). In contrast, germ cells show high frequency of HR caused by programmed DSBs. Microsatellites are prone to DSBs during genome replication and, thereby, capable of promoting HR. It remains unclear whether HR occurs frequently at microsatellites both in normal somatic cells and germ cells in a similar manner. Results By examining the linkage pattern of multiple paternal and maternal markers flanking innate GT microsatellites, we measured HR at the GT microsatellites in various somatic cells and germ cells in a goldfish intraspecific heterozygote. During embryogenesis, the HR products accumulate gradually with the increase of the number of cell divisions. The frequency of HR at the GT microsatellites in advanced embryos, adult tissues and germ cells is surprisingly high. The type of exchanges between the homologous chromosomes is similar in normal advanced embryos and germ cells. Furthermore, a long GT microsatellite is more active than a short one in promoting HR in both somatic and germ cells. Conclusions HR occurs frequently at innate GT microsatellites in normal somatic cells and germ cells in a similar manner.

Abstract Background Renal cell carcinoma accounts for 2–3% of all cancers and metastasis increased the malignancy of renal cancer. However, the role of methylation in metastasis of renal cancer is poorly understood. Methods We performed targeted gene array to compare the differential expressions of methylation regulated genes in metastatic and primary renal cancer tissues. Quantitative methylation specific PCR was performed to examine the CpG methylation levels of Runt related transcription factor 3 (RUNX3) and transforming growth factor (TGF)-β. Western blot was performed to detect the expression of target genes. Murine xenograft renal cancer model was established to assay gene expression, methylation level, tumor growth and animal survival in vivo. Results RUNX3 and TGF-β levels were decreased in metastatic renal cancer tissues as a result of their CpG methylation. Metastatic xenograft model displayed decreased expression levels of RUNX3 and TGF-β and higher CpG methylation levels, bigger tumor size and shorter survival time, all which were restored by treatment with a methylation inhibitor. Conclusions Hypermethylation in CpG islands promotes metastasis of renal cancer and is associated with TGF-β and RUNX3 inhibition.

At present, artificial intelligence (AI) has made considerable progress in recognition of speech, face, and emotion. Potential application to robots could bring significant improvement on intelligent robotic systems. However, limited resource on robots cannot satisfy the large-scale computation and storage that the AI recognition requires. Cloud provides an efficient way for robots, where they off-load the computation too. Therefore, we present a cognition-based context-aware cloud computing framework, which is designed to help robot's sense environments including user's emotions. Based on the recognized context information, robots could optimize their responses and improve the user's experience on interaction. The framework contains a customizable context monitoring system on the mobile end to collect and process the data from the robot's sensors. Besides, it integrates various AI recognition services in the cloud to extract the context facts by analyzing and understanding the data. Once the context data is extracted, the results are pushed back to mobile end for making a better decision in the next interactions. In this paper, we demonstrate and evaluate the framework by a real case, an educational mobile app for English learning. The results show that the proposed framework could significantly improve the interaction and intelligence of mobile robots.

In the spinal cord, excitatory V2a and inhibitory V2b interneurons are produced together by the final division of common P2 progenitors. During V2a and V2b diversification, Tal1 is necessary and sufficient to promote V2b differentiation and Vsx2 suppresses the expression of motor neuron genes to consolidate V2a interneuron identity. The expression program of Tal1 is triggered by a Foxn4-driven regulatory network in the common P2 progenitors. Why the expression of Tal1 is inhibited in V2a interneurons at the onset of V2a and V2b sub-lineage diversification remains unclear. Since transcription repressor Vsx1 is expressed in the P2 progenitors and newborn V2a cells in zebrafish, we investigated the role of Vsx1 in V2a fate specification during V2a and V2b interneuron diversification in this species by loss and gain-of-function experiments. In vsx1 knockdown embryos or knockout Go chimeric embryos, tal1 was ectopically expressed in the presumptive V2a cells, while the generation of V2a interneurons was significantly suppressed. By contrast, in vsx1 overexpression embryos, normal expression of tal1 in the presumptive V2b cells was suppressed, while the generation of V2a interneuron was expanded. Chromatin immunoprecipitation and electrophoretic mobility shift assays in combination with core consensus sequence mutation analysis further revealed that Vsx1 can directly bind to tal1 promoter and repress tal1 transcription. These results indicate that Vsx1 can directly repress tal1 transcription and plays an essential role in defining V2a interneuron sub-lineage during V2a and V2b sub-lineage diversification in zebrafish.

It is now widely accepted that allele-specific DNA methylation (ASM) commonly occurs at non-imprinted loci. Most of the non-imprinted ASM regions observed both within and outside of the CpG island show a strong correlation with DNA polymorphisms. However, what polymorphic cis-acting elements mediate non-imprinted ASM of the CpG island remains unclear. In this study, we investigated the impact of polymorphic GT microsatellites within the gene promoter on non-imprinted ASM of the local CpG island in goldfish. We generated various goldfish heterozygotes, in which the length of GT microsatellites or some non-repetitive sequences in the promoter of no tail alleles was different. By examining the methylation status of the downstream CpG island in these heterozygotes, we found that polymorphisms of a long GT microsatellite can lead to the ASM of the downstream CpG island during oogenesis and embryogenesis, polymorphisms of short GT microsatellites and non-repetitive sequences in the promoter exhibited no significant effect on the methylation of the CpG island. We also observed that the ASM of the CpG island was associated with allele-specific expression in heterozygous embryos. These results suggest that a long polymorphic GT microsatellite within a gene promoter mediates non-imprinted ASM of the local CpG island in a goldfish inter-strain hybrid.

Accumulating evidence indicates that oxidative stress plays a critical role in initiating the progression of inflammatory and fibrotic liver diseases, including cholestatic hepatitis. Peroxiredoxin 4 (PRDX4) is a secretory antioxidase that protects against oxidative damage by scavenging reactive oxygen species (ROS) in both the intracellular compartments and extracellular space. In this study, we examined the in vivo net effects of PRDX4 overexpression in a murine model of cholestasis. To induce cholestatic liver injury, we subjected C57BL/6J wild-type (WT) or human PRDX4 (hPRDX4) transgenic (Tg) mice to sham or bile duct ligation (BDL) surgery for seven days. Our results showed that the liver necrosis area was significantly suppressed in Tg BDL mice with a reduction in the severity of liver injuries. Furthermore, PRDX4 overexpression markedly reduced local and systemic oxidative stress generated by BDL. In addition, suppression of inflammatory cell infiltration, reduced proliferation of hepatocytes and intrahepatic bile ducts, and less fibrosis were also found in the liver of Tg BDL mice, along with a reduced mortality rate after BDL surgery. Interestingly, the composition of the hepatic bile acids (BAs) was more beneficial for Tg BDL mice than for WT BDL mice, suggesting that PRDX4 overexpression may affect BA metabolism during cholestasis. These features indicate that PRDX4 plays an important role in protecting against liver injury following BDL and might be a promising therapeutic modality for cholestatic diseases.

Tandem repeats (TRs) are abundant and widely distributed in eukaryotic genomes. TRs are thought to have various functions in gene transcription, DNA methylation, nucleosome position and chromatin organization. Variation of repeat units in the genome is observed in association with a number of diseases, such as Fragile X Syndrome, Huntington's disease and Friedreich's ataxia. However, the underlying mechanisms involved are poorly understood, largely owing to the technical limitations in modification of TRs at definite sites in the genome in vivo. Transcription activator-like effector nucleases (TALENs) are widely used in recent years in gene targeting for their specific binding to target sequences when engineered in vitro. Here, we show that the repair of a double-strand break (DSB) induced by TALENs adjacent to a TR can produce serial types of mutations in the TR region. Sequencing analysis revealed that there are three types of mutations induced by the DSB repair, including indels only within the TR region or within the flanking TALEN target region or simutaneously within both regions. Therefore, desired TR mutant types can be conveniently obtained by using engineered TALENs. These results demonstrate that TALENs can serve as a convenient tool for modifying TRs in the genome in studying the functions of TRs.