CRISPR/Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. High-fidelity Cas9 variants have been identified; however, they often have reduced activity, constraining their utility, which presents a major challenge for their use in research applications and therapeutics. Here we developed a tRNAGln-processing system to restore the activity of multiple high-fidelity Cas9 variants in human cells, including SpCas9-HF1, eSpCas9, and xCas9. Specifically, acting on previous observations that small guide RNAs (sgRNAs) harboring an extra A or G (A/G) in the first 5′ nucleotide greatly affect the activity of high-fidelity Cas9 variants and that tRNA–sgRNA fusions improve Cas9 activity, we investigated whether a GN20 sgRNA fused to different tRNAs (G-tRNA-N20) could restore the activity of SpCas9 variants in human cells. Using flow cytometry, a T7E1 assay, deep sequencing–based DNA cleavage activity assays, and HEK-293 cells, we observed that a tRNAGln–sgRNA fusion system enhanced the activity of Cas9 variants, which could be harnessed for efficient correction of a pathogenic mutation in the retinoschisin 1 (RS1) gene, resulting in 6- to 8-fold improved Cas9 activity. We propose that the tRNA-processing system developed here specifically for human cells could facilitate high-fidelity Cas9-mediated human genome-editing applications. CRISPR/Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. High-fidelity Cas9 variants have been identified; however, they often have reduced activity, constraining their utility, which presents a major challenge for their use in research applications and therapeutics. Here we developed a tRNAGln-processing system to restore the activity of multiple high-fidelity Cas9 variants in human cells, including SpCas9-HF1, eSpCas9, and xCas9. Specifically, acting on previous observations that small guide RNAs (sgRNAs) harboring an extra A or G (A/G) in the first 5′ nucleotide greatly affect the activity of high-fidelity Cas9 variants and that tRNA–sgRNA fusions improve Cas9 activity, we investigated whether a GN20 sgRNA fused to different tRNAs (G-tRNA-N20) could restore the activity of SpCas9 variants in human cells. Using flow cytometry, a T7E1 assay, deep sequencing–based DNA cleavage activity assays, and HEK-293 cells, we observed that a tRNAGln–sgRNA fusion system enhanced the activity of Cas9 variants, which could be harnessed for efficient correction of a pathogenic mutation in the retinoschisin 1 (RS1) gene, resulting in 6- to 8-fold improved Cas9 activity. We propose that the tRNA-processing system developed here specifically for human cells could facilitate high-fidelity Cas9-mediated human genome-editing applications.
Cpf1 has been harnessed as a tool for genome manipulation in various species because of its simplicity and high efficiency. Our recent study demonstrated that FnCpf1 could be utilized for human genome editing with notable advantages for target sequence selection due to the flexibility of the protospacer adjacent motif (PAM) sequence. Multiplex genome editing provides a powerful tool for targeting members of multigene families, dissecting gene networks, modeling multigenic disorders in vivo, and applying gene therapy. However, there are no reports at present that show FnCpf1-mediated multiplex genome editing via a single customized CRISPR RNA (crRNA) array. In the present study, we utilize a single customized crRNA array to simultaneously target multiple genes in human cells. In addition, we also demonstrate that a single customized crRNA array to target multiple sites in one gene could be achieved. Collectively, FnCpf1, a powerful genome-editing tool for multiple genomic targets, can be harnessed for effective manipulation of the human genome. Cpf1 has been harnessed as a tool for genome manipulation in various species because of its simplicity and high efficiency. Our recent study demonstrated that FnCpf1 could be utilized for human genome editing with notable advantages for target sequence selection due to the flexibility of the protospacer adjacent motif (PAM) sequence. Multiplex genome editing provides a powerful tool for targeting members of multigene families, dissecting gene networks, modeling multigenic disorders in vivo, and applying gene therapy. However, there are no reports at present that show FnCpf1-mediated multiplex genome editing via a single customized CRISPR RNA (crRNA) array. In the present study, we utilize a single customized crRNA array to simultaneously target multiple genes in human cells. In addition, we also demonstrate that a single customized crRNA array to target multiple sites in one gene could be achieved. Collectively, FnCpf1, a powerful genome-editing tool for multiple genomic targets, can be harnessed for effective manipulation of the human genome.
This study was aimed to evaluate effects of dietary protein levels on the nursing performance of lactating mink (Neovison vison).A total of 120 pregnant mink were randomly assigned to 4 dietary treatment groups with protein levels of 32% (P32, control), 36% (P36), 40% (P40) and 44% (P44) (dry matter basis, corresponding to 29%, 32%, 36% and 39% of ME), and fed ad libitum for 8 weeks from late gestation to the end of lactation.Results showed that daily weight gain of kits increased linearly with the increasing dietary protein levels although the difference was not significant.Similar trend was noted for the survival rate of kits at weaning.Dams fed diets with higher protein levels showed less body weight loss during lactation.Improved metabolic status was demonstrated by the changed metabolic profiles as indicated blood urea nitrogen level (P<0.05),total protein (P<0.05),glutamate pyruvate transaminase activity and blood glucose content (P<0.05).In conclusion, we found that dietary protein intake significantly affects the nursing performance of female mink.We suggest that an estimated dietary protein level of 44% (39% of ME) could be used as a guidance to achieve the optimal performance of lactating dams.
Abstract Familial hypercholesterolemia (FH) is one of the most prevalent monogenetic disorders leading to cardiovascular disease (CVD) worldwide. Mutations in Ldlr , encoding a membrane‐spanning protein, account for the majority of FH cases. No effective and safe clinical treatments are available for FH. Adenine base editor (ABE)‐mediated molecular therapy is a promising therapeutic strategy to treat genetic diseases caused by point mutations, with evidence of successful treatment in mouse disease models. However, due to the differences in the genomes between mice and humans, ABE with specific sgRNA, a key gene correction component, cannot be directly used to treat FH patients. Thus, we generated a knock‐in mouse model harboring the partial patient‐specific fragment and including the Ldlr W490X mutation. Ldlr W490X/W490X mice recapitulated cholesterol metabolic disorder and clinical manifestations of atherosclerosis associated with FH patients, including high plasma low‐density lipoprotein cholesterol levels and lipid deposition in aortic vessels. Additionally, we showed that the mutant Ldlr gene could be repaired using ABE with the cellular model. Taken together, these results pave the way for ABE‐mediated molecular therapy for FH.
Summary The objective of this study was to determine whether nutrient digestibility and production performance of growing‐furring blue foxes ( Alopex lagopus ) are affected by different dietary fat levels. Sixty‐four young animals were randomly assigned to four groups (A, B, C, D) provided with diets containing approximately 12%, 26%, 40%, 54% fat in the dry matter respectively. When dietary fat level was increased, the apparent digestibility of main nutrients except for crude carbohydrates, and gross energy were improved (p < 0.0001). The amount of nitrogen excreted was reduced and the biological value of protein was enhanced (p < 0.0001). Over the experimental phase, the efficiency of metabolizable energy (ME) used for gain in group B (26% fat content) was higher than that in other groups. When diets with 12–26% fat content were fed, there was an increasing tendency in skin size. But it had negative effects on skin size and fur quality when the amount of fat content was over 40%. In conclusion, the experiment showed that dietary fat could significantly improve some nutrient utilization and significantly reduce feed/gain ratio as a main energy source. The most preferable fur quality and efficiency of ME used for gain were obtained when diet contained 26% fat level in growing‐furring period.
A completely randomized 3 × 3 + 1 factorial experiment was conducted to evaluate the effects of sources and concentrations of Zn on growth performance, nutrient digestibility, serum biochemical endpoints, and fur quality in growing-furring female black mink. One hundred fifty healthy 15-wk-old female mink were randomly allocated to 10 dietary treatments ( = 15/group) for a 60-d trial. Animals in the control group were fed a basal diet, which consisted of mainly corn, soybean oil, meat and bone meal, and fish meal, with no Zn supplementation. Mink in the other 9 treatments were fed the basal diet supplemented with Zn from either zinc sulfate (ZnSO), zinc glycinate (ZnGly), or Zn pectin oligosaccharides (ZnPOS) at concentrations of either 100, 300, or 900 mg Zn/kg DM. The results showed that mink in the ZnPOS groups had higher ADG than those in the ZnSO groups (main effect, < 0.05). The addition of Zn reduced the G:F ( < 0.05). In addition, CP and crude fat digestibility were linearly increased with Zn supplementation ( < 0.05) and N retention tended to increase with Zn addition ( = 0.08). Dietary Zn supplementation increased the concentration of serum albumin and activity of alkaline phosphatase ( < 0.05). There was a linear effect of dietary Zn on the concentration of tibia Zn and pancreatic Zn ( < 0.05). For fur quality characteristics, the fur density and hair color of mink were improved by dietary Zn concentration ( < 0.05). Compared with ZnSO (100%), relative bioavailability values of ZnGly were 115 and 118%, based on tibia and pancreatic Zn, respectively, and relative bioavailability values of ZnPOS were 152 and 142%, respectively. In conclusion, this study demonstrates that Zn supplementation can promote growth and increase nutrient digestibility and fur quality and that ZnPOS is more bioavailable than ZnSO and ZnGly in growing-furring female mink.
Abstract The substation is an important node connecting the backbone network and distribution network and is the core hub of the smart grid. With the continuous increase of electrical equipment, the substation plays a vital role in the whole power system. The structural principle of deep learning convolutional neural network is deeply studied, which lays a theoretical foundation for substation fault detection and recognition based on the object detection algorithm. This paper focuses on fault detection algorithms based on deep learning. First, two mainstream target detection algorithms, Faster R-CNN and YOLO V5, are applied to the self-made substation fault data set. The experimental comparison shows that while the YOLO V5 algorithm has a slightly higher recognition speed, the accuracy of fault target recognition and similar background image recognition is higher than that of the Faster R-CNN algorithm. Then, for the benchmark network YOLO V5, an improved method is proposed. The innovative idea is to delete large branches from the network detection part for the detection of small faults in the station, so as to reduce the number of model parameters and improve the speed of model detection.
An experiment was carried out to examine the effects of low-protein diets supplemented with different levels of DL-methionine (Met) and Lysine (Lys) on growth performance of growing-furring blue foxes in order to find the optimal dietary supplementation levels of Met and Lys. For two protein levels, conventional 27% (P27) and low 19% (P19) on dry matter basis, respectively, and the low-protein diets were supplemented with Met (0.3%, 0.5%, 0.7%) and Lys (0.4%, 0.6%, 0.8%). An entirely random experimental design was adopted with two factors (3×3) and totally 10 groups (P27, L1M1, L1M2, L1M3, L2M1, L2M2, L2M3, L3M1, L3M2 and L3M3). From mid-September to pelting, based on the average daily gain, daily N retention, N retention ratio and the performance of blue foxes in different groups, 0.6% Met supplementation in low-protein diet was optimum; based on the daily N retention, N biological value and the quality of the fur, 0.3% and 0.5% Lys supplementation were optimum; based on the N apparent digestibility and daily N output, 0.3% Lys supplementation was optimum. In this experiment, the performance of blue foxes in L1M2 0.3% Lys×0.6% Met group was better than that in the other groups, which indicates that low-protein diets supplemented with DL-methionine and lysine for blue foxes can be beneficial to reduce feed expenses and nitrogen emission to the environment.
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