Precise large-fragment deletions in mammalian cells and mice generated by dCas9-controlled CRISPR/Cas3.

Currently, the Cas9 and Cas12a systems are widely used for genome editing, but their ability to precisely generate large chromosome fragment deletions is limited. Type I-E CRISPR mediates broad and unidirectional DNA degradation, but controlling the size of Cas3-mediated DNA deletions has proven elusive thus far. Here, we demonstrate that the endonuclease deactivation of Cas9 (dCas9) can precisely control Cas3-mediated large-fragment deletions in mammalian cells. In addition, we report the elimination of the Y chromosome and precise retention of the Sry gene in mice using CRISPR/Cas3 and dCas9-controlled CRISPR/Cas3, respectively. In conclusion, dCas9-controlled CRISPR/Cas3-mediated precise large-fragment deletion provides an approach for establishing animal models by chromosome elimination. This method also holds promise as a potential therapeutic strategy for treating fragment mutations or human aneuploidy diseases that involve additional chromosomes.

The Influence of B4C Film Density on Damage Threshold Based on Monte Carlo Method for X-ray Mirror.

The uniformity and consistency of X-ray mirror film materials prepared by experimental methods are difficult to guarantee completely. These factors directly affect the service life of free electron laser devices in addition to its own optical properties. Therefore, the quality of the film material, especially the density, has a critical effect on its application. Boron carbide film and monocrystalline silicon substrate were suitable examples to explore their influence of density on the damage threshold based on Monte Carlo and heat-conduction methods. Through simulation results, it was found that the change in film density could affect the energy deposition depth and damage threshold. When the film density was 2.48 g/cm3, it had relatively high damage threshold in all energy ranges. And then the specific incident parameter for practical application was investigated. It was found that the damage mechanism of the B4C/Si was the melting of the interface. And the damage threshold was also higher with the film density of 2.48 g/cm3. Therefore, it was recommended to maintain the density at this value as far as possible when preparing the film, and to ensure the uniformity and consistency of the film material.

Effect of Comprehensive Nursing on Pain Relief, Comfort and Burden of Family Care of Infantile Anal Fistula.

Objective: To explore the effect of comprehensive nursing on pain relief, comfort and burden of family care of infantile anal fistula. Methods: This was a randomized, double-blind, controlled clinical trial. A total of 106 cases of children with anal fistula from January 2021 to December 2021 were selected and divided into observation group and control group, there were 53 cases in each group. The control group were underwent with routine nursing intervention. The observation group were underwent with video health education, peer support, music relaxation training, physiotherapy and auricular point pressing on the basis of routine nursing therapy and other measures (comprehensive nursing). The wound healing time, comfort score, complication rate and family care burden of two groups were compared. Results: After intervention, the scores of pain degree in the observation group were significantly lower than those in the control group (4.02 ± 0.85 vs 5.89 ± 2.36, p < 0.05), and the scores of comfort degree in the observation group were higher than those in the control group (70.23 ± 5.98 vs 46.88 ± 5.23, p < 0.05). After intervention, the wound healing time of the observation group was shorter than that of the control group (3.98 ± 0.63 vs 5.77 ± 1.02, p < 0.05), and the crying times of the observation group were less than that of the control group (1.22 ± 0.26 vs 4.02 ± 0.48, p < 0.05). After intervention, the total scores of family members' care load in the observation group were significantly lower than those in the control group (37.26 ± 4.78 vs 55.99 ± 5.02, p < 0.05). Conclusion: Comprehensive nursing can effectively promote wound healing in infantile anal fistula, reduce pain, prompt children's comfort, reduce the number of children crying, and reduce the burden of care for children's families.

Programmable RNA 5-methylcytosine (m5C) modification of cellular RNAs by dCasRx conjugated methyltransferase and demethylase.

5-Methylcytosine (m5C), an abundant RNA modification, plays a crucial role in regulating RNA fate and gene expression. While recent progress has been made in understanding the biological roles of m5C, the inability to introduce m5C at specific sites within transcripts has hindered efforts to elucidate direct links between specific m5C and phenotypic outcomes. Here, we developed a CRISPR-Cas13d-based tool, named reengineered m5C modification system (termed 'RCMS'), for targeted m5C methylation and demethylation in specific transcripts. The RCMS editors consist of a nuclear-localized dCasRx conjugated to either a methyltransferase, NSUN2/NSUN6, or a demethylase, the catalytic domain of mouse Tet2 (ten-eleven translocation 2), enabling the manipulation of methylation events at precise m5C sites. We demonstrate that the RCMS editors can direct site-specific m5C incorporation and demethylation. Furthermore, we confirm their effectiveness in modulating m5C levels within transfer RNAs and their ability to induce changes in transcript abundance and cell proliferation through m5C-mediated mechanisms. These findings collectively establish RCMS editors as a focused epitranscriptome engineering tool, facilitating the identification of individual m5C alterations and their consequential effects.

Targeted mutagenesis in mice via an engineered AsCas12f1 system.

SpCas9 and AsCas12a are widely utilized as genome editing tools in human cells, but their applications are largely limited by their bulky size. Recently, AsCas12f1 protein, with a small size (422 amino acids), has been demonstrated to be capable of cleaving double-stranded DNA protospacer adjacent motif (PAM). However, low editing efficiency and large differences in activity against different genomic loci have been a limitation in its application. Here, we show that engineered AsCas12f1 sgRNA has significantly improved the editing efficiency in human cells and mouse embryos. Moreover, we successfully generated three stable mouse mutant disease models using the engineered CRISPR-AsCas12f1 system in this study. Collectively, our work uncovers the engineered AsCas12f1 system expands mini CRISPR toolbox, providing a remarkable promise for therapeutic applications.

Exploring moral algorithm preferences in autonomous vehicle dilemmas: an empirical study.

Introduction: This study delves into the ethical dimensions surrounding autonomous vehicles (AVs), with a specific focus on decision-making algorithms. Termed the "Trolley problem," an ethical quandary arises, necessitating the formulation of moral algorithms grounded in ethical principles. To address this issue, an online survey was conducted with 460 participants in China, comprising 237 females and 223 males, spanning ages 18 to 70. Methods: Adapted from Joshua Greene's trolley dilemma survey, our study employed Yes/No options to probe participants' choices and Likert scales to gauge moral acceptance. The primary objective was to assess participants' inclinations toward four distinct algorithmic strategies-Utilitarianism, Rawlsianism, Egoism, and a Hybrid approach-in scenarios involving AVs. Results: Our findings revealed a significant disparity between participants' preferences in scenarios related to AV design and those focused on purchase decisions. Notably, over half of the respondents expressed reluctance to purchase AVs equipped with an "egoism" algorithm, which prioritizes the car owner's safety. Intriguingly, the rejection rate for "egoism" was similar to that of "utilitarianism," which may necessitate self-sacrifice. Discussion: The hybrid approach, integrating "Utilitarianism" and "Egoism," garnered the highest endorsement. This highlights the importance of balancing self-sacrifice and harm minimization in AV moral algorithms. The study's insights are crucial for ethically and practically advancing AV technology in the continually evolving realm of autonomous vehicles.

A strategy for Cas13 miniaturization based on the structure and AlphaFold.

The small size of the Cas nuclease fused with various effector domains enables a broad range of function. Although there are several ways of reducing the size of the Cas nuclease complex, no efficient or generalizable method has been demonstrated to achieve protein miniaturization. In this study, we establish an Interaction, Dynamics and Conservation (IDC) strategy for protein miniaturization and generate five compact variants of Cas13 with full RNA binding and cleavage activity comparable the wild-type enzymes based on a combination of IDC strategy and AlphaFold2. In addition, we construct an RNA base editor, mini-Vx, and a single AAV (adeno-associated virus) carrying a mini-RfxCas13d and crRNA expression cassette, which individually shows efficient conversion rate and RNA-knockdown activity. In summary, these findings highlight a feasible strategy for generating downsized CRISPR/Cas13 systems based on structure predicted by AlphaFold2, enabling targeted degradation of RNAs and RNA editing for basic research and therapeutic applications.

Constructing abundant interfaces by decorating MoP quantum dots on CoP nanowires to induce electronic structure modulation for enhanced hydrogen evolution reaction.

Interface engineering is a method of enhancing catalytic activity while maintaining a material's surface properties. Thus, we explored the interface effect mechanism via a hierarchical structure of MoP/CoP/Cu3P/CF. Remarkably, the heterostructure MoP/CoP/Cu3P/CF demonstrates an outstanding overpotential of 64.6 mV at 10 mA cm-2 with a Tafel slope of 68.2 mV dec-1 in 1 M KOH. DFT calculations indicate that the MoP/CoP interface in the catalyst exhibited the most favorable H* adsorption characteristics (-0.08 eV) compared to the pure phases of CoP (0.55 eV) and MoP (0.22 eV). This result can be attributed to the apparent modulation of electronic structures within the interface domains. Additionally, the CoCH/Cu(OH)2/CF‖MoP/CoP/Cu3P/CF electrolyzer demonstrates excellent overall water splitting performance, achieving 10 mA cm-2 in 1 M KOH solution with a modest voltage of only 1.53 V. This electronic structure adjustment via interface effects provides a new and efficient approach to prepare high-performance hydrogen production catalysts.

Development of Woolly Hair and Hairlessness in a CRISPR-Engineered Mutant Mouse Model with KRT71 Mutations.

Hypotrichosis simplex (HS) and woolly hair (WH) are rare and monogenic disorders of hair loss. HS, characterized by a diffuse loss of hair, usually begins in early childhood and progresses into adulthood. WH displays strong coiled hair involving a localized area of the scalp or covering the entire side. Mutations in the keratin K71(KRT71) gene have been reported to underlie HS and WH. Here, we report the generation of a mouse model of HS and WH by the co-injection of Cas9 mRNA and sgRNA, targeting exon6 into mouse zygotes. The Krt71-knockout (KO) mice displayed the typical phenotypes, including Krt71 protein expression deletion and curly hair in their full body. Moreover, we found that mice in 3-5 weeks showed a new phenomenon of the complete shedding of hair, which was similar to nude mice. However, we discovered that the mice exhibited no immune deficiency, which was a typical feature of nude mice. To our knowledge, this novel mouse model generated by the CRISPR/Cas9 system mimicked woolly hair and could be valuable for hair disorder studies.

Unique progerin C-terminal peptide ameliorates Hutchinson-Gilford progeria syndrome phenotype by rescuing BUBR1.

An accumulating body of evidence indicates an association between mitotic defects and the aging process in Hutchinson-Gilford progeria syndrome (HGPS), which is a premature aging disease caused by progerin accumulation. Here, we found that BUBR1, a core component of the spindle assembly checkpoint, was downregulated during HGPS cellular senescence. The remaining BUBR1 was anchored to the nuclear membrane by binding with the C terminus of progerin, thus further limiting the function of BUBR1. Based on this, we established a unique progerin C-terminal peptide (UPCP) that effectively blocked the binding of progerin and BUBR1 and enhanced the expression of BUBR1 by interfering with the interaction between PTBP1 and progerin. Finally, UPCP significantly inhibited HGPS cellular senescence and ameliorated progeroid phenotypes, extending the lifespan of LmnaG609G/G609G mice. Our findings reveal an essential role for the progerin-PTBP1-BUBR1 axis in HGPS. Therapeutics designed around UPCP may be a beneficial strategy for HGPS treatment.

Anti-hsa-miR-59 alleviates premature senescence associated with Hutchinson-Gilford progeria syndrome in mice.

Hutchinson-Gilford progeria syndrome (HGPS) is a lethal premature aging disorder without an effective therapeutic regimen. Because of their targetability and influence on gene expression, microRNAs (miRNAs) are attractive therapeutic tools to treat diseases. Here we identified that hsa-miR-59 (miR-59) was markedly upregulated in HGPS patient cells and in multiple tissues of an HGPS mouse model (LmnaG609G/G609G ), which disturbed the interaction between RNAPII and TFIIH, resulting in abnormal expression of cell cycle genes by targeting high-mobility group A family HMGA1 and HMGA2. Functional inhibition of miR-59 alleviated the cellular senescence phenotype of HGPS cells. Treatment with AAV9-mediated anti-miR-59 reduced fibrosis in the quadriceps muscle, heart, and aorta, suppressed epidermal thinning and dermal fat loss, and yielded a 25.5% increase in longevity of LmnaG609G/G609G mice. These results identify a new strategy for the treatment of HGPS and provide insight into the etiology of HGPS disease.

A Very Fast Image Stitching Algorithm for PET Bottle Caps.

In the beverage, food and drug industry, more and more machine vision systems are being used for the defect detection of Polyethylene Terephthalate (PET) bottle caps. In this paper, in order to address the result of cylindrical distortions that influence the subsequent defect detection in the imaging process, a very fast image stitching algorithm is proposed to generate a panorama planar image of the surface of PET bottle caps. Firstly, the three-dimensional model of the bottle cap is established. Secondly, the relative poses among the four cameras and the bottle cap in the three-dimensional space are calculated to obtain the mapping relationship between three-dimensional points on the side surface of the bottle cap and image pixels taken by the camera. Finally, the side images of the bottle cap are unfolded and stitched to generate a planar image. The experimental results demonstrate that the proposed algorithm unfolds the side images of the bottle cap correctly and very fast. The average unfolding and stitching time for 1.6-megapixel color caps image can reach almost 123.6 ms.

Fucosyltransferase 2: A Genetic Risk Factor for Intestinal Diseases.

The fucosyltransferase 2 gene (FUT2) mediates the synthesis of histoblood group antigens (HBGA) that occur in vivo from multiple organs, particularly on the surface of intestinal epithelial cells and body fluids. To date, many studies have demonstrated that the interaction of HBGA with the host microbiota is the cause of pathogenesis of intestinal diseases, making FUT2 non-secretor a risk factor for inflammatory bowel disease (IBD) due to the lack of HBGA. As HBGA also acts as an attachment site for norovirus (NoV) and rotavirus (RV), the non-secretor becomes a protective factor for both viral infections. In addition, the interaction of norovirus and rotavirus with symbiotic bacteria has been found to play an important role in regulating enteroviral infection in IBD. Given the current incomplete understanding of the complex phenomenon and the underlying pathogenesis of intestinal diseases such as IBD, it has recently been hypothesized that the FUT2 gene regulates intestinal bacteria through attachment sites, may help to unravel the role of FUT2 and intestinal flora in the mechanism of intestinal diseases in the future, and provide new ideas for the prevention and treatment of intestinal diseases through more in-depth studies.

Live imaging of RNA and RNA splicing in mammalian cells via the dcas13a-SunTag-BiFC system.

Dynamic tracking of the localization of RNA molecules (nucleus and/or cytoplasm) and RNA splicing in living cells plays an important role in understanding their functions. However, a lack of dynamic imaging and high background fluorescence have been reported in the fluorescence in situ hybridization (FISH). Here, we developed a new tool, the dcas13a-SunTag-BiFC system, which fused the dLwacas13a and SunTag systems. dLwacas13a is used as a tracker to target specific RNAs, while SunTag recruits split Venus fluorescent proteins to label targeted RNAs. Our results showed that 4 × NLS-dCas13a-24 × SunTag-BiFC and 2 × NLS- dCas13a-24 × SunTag-BiFC systems can be used for imaging of endogenous RNA foci in the nucleus (Xist) and cytoplasm (Ppib and stress granules) in living cells, respectively. Compared to 12x MS2-MCP system, the dcas13a-SunTag-BiFC system showed a better performance of mRNA foci tracking in live cells. Furthermore, we confirmed the premature termination codon (PTC)-induced exon skipping of Oxt RNA using the dcas13a-SunTag-BiFC and MS2-MCP systems in the nucleus. Thus, the dcas13a-SunTag-BiFC system will facilitate the study of RNA localization in living cells and provide new insights into RNA translocation and splicing.

No apparent p53 activation in CRISPR-engineered gene-edited rabbits.

Clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9) and base editors (BEs) are revolutionary gene-editing technology that has been widely utilized in biology, biotechnology and medicine. However, recent reports show that CRISPR-Cas9-mediated genome editing can induce a p53-mediated stress response and cell cycle arrest in human cells, while not illustrated in gene-editing animals. In the study, to verify whether there is a phenomenon of p53 activation, by analysing nine gene-edited rabbits using CRISPR-Cas9 and BEs, we provide the first evidence that no apparent p53 expression changes in those rabbits generated by Cas9 or BE-edited, suggesting that p53 may not need to consider for application in gene-edited animals.

Reduced off-target effect of NG-BE4max by using NG-HiFi system.

Recently, a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize a minimal NG protospacer adjacent motif (PAM) was reported to expand the targeting scope in genome editing. However, increased genome-wide off-target mutations with this variant compared with SpCas9 were reported in previous studies. In addition, lower base editing frequencies and higher unintended off-target mutations were also found in Hoxc13-ablated rabbits generated by NG-BE4max in our study. Here, a high-fidelity base editor, NG-HiFi, in comparison to NG-BE4max, showed retention of on-target activity while exhibiting significantly decreased off-target activity in Hoxc13-ablated rabbits. Collectively, the improved specificity and reduced off-target effect of SpCas9-NG assisted in cytidine base editing with the NG-HiFi system, providing a promising tool to precisely model human diseases in rabbits.

Study on burgers vector of dislocations in KDP (010) faces and screw dislocation growth mechanism of (101) faces.

We modified the conventional etching-optical method to measure dislocation direction in a KDP crystal. As burgers vector of dislocation in the KDP crystal must match the minimum periodic vector of the crystal lattice, we suggest that dislocations with a burgers vector of [101], [102] and [103] exist. Atomic force microscopy was employed to characterize the morphology of growth spirals on the hillock of (101) faces. Multi-spirals consisting of more than two element steps with a height of 0.5 nm which is equal to (101) face interplanar distances were observed. We propose the multi-spiral structure is determined by the burgers vector of the corresponding dislocation, and constructed a geometric model of the crystal with screw dislocation to derive the relationship. Growth spirals on the (101) face present a particular triangular morphology and we proved that the triangle structure is formed by connected steps in the 1/2[111] and [010] direction. Micropipes form when the magnitude of the dislocation's burgers vector exceeds 1 nm, as predicted by BCF theory. Interaction between dislocations was observed also.